Collagen-Hydroxyapatite Scaffolds Induce Human Adipose Derived Stem Cells Osteogenic Differentiation In Vitro

Abstract

Mesenchymal stem cells (MSCs) play a crucial role in regulating normal skeletal homeostasis and, in case of injury, in bone healing and reestablishment of skeletal integrity. Recent scientific literature is focused on the development of bone regeneration models where MSCs are combined with biomimetic three-dimensional scaffolds able to direct MSC osteogenesis. In this work the osteogenic potential of human MSCs isolated from adipose tissue (hADSCs) has been evaluated in vitro in combination with collagen/Mg doped hydroxyapatite scaffolds. Results demonstrate the high osteogenic potential of hADSCs when cultured in specific differentiation induction medium, as revealed by the Alizarin Red S staining and gene expression profile analysis. In combination with collagen/hydroxyapatite scaffold, hADSCs differentiate into mature osteoblasts even in the absence of specific inducing factors; nevertheless, the supplement of the factors markedly accelerates the osteogenic process, as confirmed by the expression of specific markers of pre-osteoblast and mature osteoblast stages, such as osterix, osteopontin (also known as bone sialoprotein I), osteocalcin and specific markers of extracellular matrix maturation and mineralization stages, such as ALPL and osteonectin. Hence, the present work demonstrates that the scaffold per se is able to induce hADSCs differentiation, while the addition of osteo-inductive factors produces a significant acceleration of the osteogenic process. This observation makes the use of our model potentially interesting in the field of regenerative medicine for the treatment of bone defects.

Fig 1. Gene expression during hADSCs osteogenic differentiation on plastic dishes.

Graphical representation of relative gene expression during 6 temporal intervals (days 0–3, 4–7, 8–11, 12–15, 16–19, 20–24) using hADSC in expansion medium as control group. qRT-PCR has been performed for the mRNA of Osteocalcin (BGLAP), Osteopontin (SPP1), ALPL, Osterix (SP7), BMP2, CDC25A, COL1A1, COL2A2, IGF2, ITGA1, MSX1, RUNX2, SOX9, TGFb1, VCAM. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Beta-Tubulin (TUBB) have been used as endogenous controls. AOV test p-value is reported and * indicates significant (p<0.05) differences between time groups and control samples as reported by the post-hoc test.

Fig 2. Representative SEM images of the scaffold bony layer.

Ultrastructural details of the bony layer are reported with three different magnification 30x, 50x and 150x.

Fig 3. Biocompatibility of biomimetic scaffold.

Haematoxylin and eosin staining of empty scaffolds maintained for 8 weeks in expansion (a) or osteogenic medium (f) and of hADSCs seeded scaffolds (b-e, g-l) maintained in the same media for 1, 2, 4 and 8 weeks, magnification 10x. (k) Graphical representation of cell count at 1, 2, 4 and 8 weeks of culture in expansion (light bars) and osteogenic medium (dark bars). Two-way ANOVA p values are reported. Symbols above bars indicate statistically significant differences (p<0.05) in the Tukey HSD post-hoc tests: 1 indicates differences with the 1 week group (same medium), 2 indicates differences with the 2 weeks group (same medium), 3 indicates differences with the 4 weeks group (same medium), 4 indicates differences with the 8 weeks group (same medium) and * indicates differences in osteogenic vs expansion medium groups (same time-points). Three independently isolated hADSC samples have been used for each time-point.

Fig 4. Matrix mineralization during osteogenesis.

Alizarin Red S staining of empty scaffolds maintained for 8 weeks in expansion (a) or osteogenic medium (f) and of hADSCs seeded scaffolds (b-e, g-j) maintained in the same media for 1, 2, 4 and 8 weeks, magnification 2.5x. (k) Graphical representation of Alizarin Red S staining resulting optical density at 1, 2, 4 and 8 weeks of culture in expansion (light bars) and osteogenic medium (dark bars) and of empty scaffolds at 8 weeks. Two-way ANOVA p values are reported. Symbols above bars indicate statistically significant differences (p<0.05) in the Tukey HSD post-hoc tests: 1 indicates differences with the empty scaffold group (same medium), 2 indicates differences with the 1 week group (same medium), 3 indicates differences with the 2 weeks group (same medium), 4 indicates differences with the 4 weeks group (same medium), 5 indicates differences with the 8 weeks group (same medium) and * indicates differences in osteogenic vs expansion medium groups (same time-points). Three independently isolated hADSC samples have been used for each time-point.

Fig 5. Expression of representative osteogenic markers.

Immunohistochemical analysis of representative osteogenic markers: (A) Osterix, (B) Osteopontin, (C) ALPL, (D) Osteonectin and (E) Osteocalcin, performed on hADSCs cultured on scaffolds either in expansion (a-d) or in osteogenic medium (e-h), at different time points (1, 2, 4 and 8 weeks), magnification 20x. (i) Average cellular positivity for osteogenic markers in both expansion (light bars) and osteogenic medium (dark bars). Two-way ANOVA p values are reported. Symbols above bars indicate statistically significant differences (p<0.05) in the Tukey HSD post-hoc tests: 1 indicates differences with the 1 week group (same medium), 2 indicates differences with the 2 weeks group (same medium), 3 indicates differences with the 4 weeks group (same medium), 4 indicates differences with the 8 weeks group (same medium) and * indicates differences in osteogenic vs expansion medium groups (same time-points). Three independently isolated hADSC samples have been used for each time-point.