Fig. 6

Prolonged cryopreservation does not reduce cell viability and bone regenerative properties of C-MSCs. Clumps of mesenchymal stem cell/extracellular matrix complexes (C-MSCs) were cryopreserved for 6 months as described in the Methods. a Left panel shows macroscopic images of cryopreserved C-MSCs (cryo-C-MSC) in a 24-well culture plate. Scale bar = 4 mm. Serial sections were stained with hematoxylin and eosin (HE) and TUNEL as indicated. In addition, to monitor the cell viability, live (green) and dead cells (red) were distinguished by using a LIVE/DEAD Viability/Cytotoxicity kit. Scale bars = 200 μm. C-MSCs cryopreserved for 6 months were transplanted into a rat calvarial defect 1.6 mm in diameter with no artificial scaffold. b Micro-CT images of bone regeneration. The calvarial defect area was scanned by micro-CT on day 28. Scale bars = 500 μm. c The volume of mineralized tissue on day 28 was quantified. Data are the mean ± SD of four rats per group. *p < 0.05, by ANOVA. d All rats were sacrificed 28 days after the implantation of cryo-C-MSCs and the calvarial bones were fixed. Coronal sections were obtained and stained with H&E. Scale bars = 500 μm. The photographs are representative of four independent experiments