A New Perspective for Bone Tissue Engineering: Human Mesenchymal Stromal Cells Well-Survive Cryopreservation on β-TCP Scaffold and Show Increased Ability for Osteogenic Differentiation

Abstract

The clinical breakthrough of bone tissue engineering (BTE) depends on the ability to provide patients routinely with BTE products of consistent pharmacological quality. The bottleneck of this approach is the availability of stem cells. To avoid this, we suggest immobilization of random-donor-derived heterologous osteoinductive MSCs onto osteoconductive matrices. Such BTE products could then be frozen and, after thawing, could be released as ready-to-use products for permanent implantation during surgery. For this purpose, we developed a simple protocol for cryopreservation of BTE constructs and evaluated the effects of this procedure on human MSC (hMSCs) metabolic and osteogenic activity in vitro. Our findings show that hMSCs can be freeze-thawed on a β-TCP scaffold through a technically simple procedure. Treated cells sustained their metabolic activity and showed favorable osteogenic potential. Mechanistically, HIF1α and YBX1 genes were activated after freeze-thawing, and supposed to be linked to enhanced osteogenesis. However, the detailed mechanisms as to how the cryopreservation procedure beneficially affects the osteogenic potential of hMSCs remains to be evaluated. Additionally, we demonstrated that our BTE products could be stored for 3 days on dry ice; this could facilitate the supply chain management of cryopreserved BTE constructs from the site of manufacture to the operating room.

Keywords: 3D culture; cryopreservation; human MSCs; osteogenic differentiation; scaffold.

Figure 1

hMSCs preseeded on scaffolds retain their metabolic activity after cryopreservation. (A) Metabolic activity of MSCs was measured 1, 2, 4, and 7 days after cryopreservation, shown in % of control cells metabolic activity. MSCs were preseeded on scaffolds 48 h prior to cryopreservation. (B) There was no significant difference in metabolic activity of hMSCs processed with two different cryopreservation protocols and measured 2 days after cryopreservation. (C) Metabolic activity of control and experimental cells at 7 and 14 days of culture in osteogenic supplemented medium showed no significant differences. (D) hMSCs were first cryopreserved in liquid nitrogen and either stored for 3 days on dry ice or stored for 3 days on dry ice and 1 day at −20 °C. The metabolic activity of cells was measured at days 2 and 7 post-thaw and is shown in % of metabolic activity of cells stored in liquid nitrogen only. (E) ALP expression is significantly enhanced in both, control and experimental group cells at day 7 of culture in osteogenic-supplemented medium, as compared to cells cultured in growth medium. (* p < 0.05, ^§ p < 0.01).

Figure 2

hMSCs distribution on a β-TCP scaffold granule before and post-thawing. Control hMSCs were seeded on a β-TCP scaffold and cultured either for 24 (A) or 72 h (B). Experimental hMSCs were seeded on a β-TCP scaffold, cultured for 24 h, frozen in liquid nitrogen for seven days, thawed, and cultured for 1 (C) and 48 (D) hours. All cells are stained with Calcein AM and DAPI. Dotted line shows the edges and pores of β-TCP granule. (5× magnification; scale bar = 100 µM).

Figure 3

Osteogenic marker genes mRNAs expression in control and experimental cells. Expression of osteogenic marker genes was evaluated in experimental and control group hMSCs at days 7 and 14 of culture in osteogenic-supplemented medium. (* p < 0.05; ** p < 0.01).

Figure 4

mRNA levels of gene expression of HIF1-α, YBX1, and VEGF and IL-6 protein secretion at days 1, 7, and 14 of culture. (A) Expression of HIF1α, YBX1, and VEGF genes was evaluated in experimental and control group hMSCs at days 1, 7, and 14 of culture in osteogenic-supplemented medium. (B) Concentration of IL-6 was measured in medium supernatants of both groups at days 1, 2, 3, and 7 of culture. (* p < 0.05).