¹Department of Trauma Surgery and Orthopedics, University Hospital, Goethe University Frankfurt, Frankfurt am Main, Deutschland

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While autologous bone grafting remains the gold standard for treating delayed and non-healing bone defects, its associated complications highlight the need for alternative approaches. Bone tissue engineering (BTE) offers a promising solution by combining bone-forming stem cells with scaffolds. However, the limited availability of mesenchymal stem cells (MSCs) presents a major obstacle, as their isolation, characterization, and expansion require substantial time and resources. To address this limitation, a pre-seeded MSC-coated scaffold would be a valuable option for clinical applications.

Objective: To assess whether BTE constructs made from pooled allogeneic MSCs, pre-seeded onto an osteoconductive matrix and cryopreserved under different conditions, can serve as a ready-to-use BTE construct for bone defect treatment.

Material and methods: Human MSCs from eight bone marrow donors were pooled, seeded onto beta-TCP scaffolds and cultured for 24 hours. The resulting MSC-coated constructs (MCCs) were frozen using an “air-dry” protocol and stored in liquid nitrogen (–196°C, 7 days; group 1). On day 7, MCCs were transferred to –20°C (1 day; group 2), –80°C (3 days; group 3), or dry ice (–80°C, 3 days; group 4). After thawing, MCCs were cultured for two weeks in growth or osteogenic medium, with fresh MSCs (without freezing/thawing, group 5) as controls. Cell metabolic activity was assessed via AlamarBlue, while osteogenic and hypoxia-related gene expression was analyzed by RT-qPCR. Bone healing was evaluated by implanting MCCs into a 5 mm critically sized femoral defect in athymic rats. Spongiosa-treated defects served as positive controls, and beta-TCP-treated defects as negative controls. After eight weeks, micro-CT, biomechanical testing, and (immuno)histological analyses were performed on the operated femurs.

Results: After thawing, MCCs stored at –20°C lost metabolic activity, whereas those stored at –80°C showed no differences from the –196°C group. Cryopreserved cells exhibited upregulated osteogenic and hypoxia-related markers compared to fresh MSCs. Histology revealed the highest new bone formation in spongiosa-treated defects (0.8±0.08), with no significant differences between fresh MSCs-treated defects and those receiving frozen/thawed MCCs (range: 0.29–0.40). CD68+ cell ratio was higher in all experimental groups vs. spongiosa, while vascularization was greatest in the –20°C and dry ice groups. Bone mineral density and mechanical strength remained comparable across all groups, with the highest mechanical strength observed in spongiosa-treated defects.

Conclusions: Freezing/thawing protocols enhanced osteogenic potential in vitro and had no negative impact on bone healing compared to freshly prepared constructs. Thus, cryopreserved MCCs hold promise for clinical applications, though spongiosa remains superior.