Use of Cryopreserved Osteogenic Matrix Cell Sheets for Bone Reconstruction

Abstract

Skeletal diseases, such as nonunion and osteonecrosis, are now treatable with tissue engineering techniques. Single cell sheets called osteogenic matrix cell sheets (OMCSs) grown from cultured bone marrow-derived mesenchymal stem cells show high osteogenic potential; however, long preparation times currently limit their clinical application. Here, we report a cryopreservation OMCS transplantation method that shortens OMCS preparation time. Cryopreserved rat OMCSs were prepared using slow-and rapid-freezing methods, thawed, and subsequently injected scaffold-free into subcutaneous sites. Rapid-and slow-frozen OMCSs were also transplanted directly to the femur bone at sites of injury. Slow-freezing resulted in higher cell viability than rapid freezing, yet all two cryopreservation methods yielded OMCSs that survived and formed bone tissue. In the rapid-and slow-freezing groups, cortical gaps were repaired and bone continuity was observed within 6 weeks of OMCS transplantation. Moreover, while no significant difference was found in osteocalcin expression between the three experimental groups, the biomechanical strength of femurs treated with slow-frozen OMCSs was significantly greater than those of non-transplant at 6 weeks post-injury. Collectively, these data suggest that slow-frozen OMCSs have superior osteogenic potential and are better suited to produce a mineralized matrix and repair sites of bone injury.

Frozen-thawed gelatin-induced osteogenic cell sheets of canine adipose-derived mesenchymal stromal cells improved fracture healing in canine model

“…Although the number of viable cells and the thickness of the layers were lower in the FT-GCSs group than in the F-GCSs group, the differences were not significant. Thawing resulted in a decrease of cellular viability of FT-GCSs ( i.e ., 76% viability compared to that of F-GCSs), which is comparable to the decrease in viability of cryopreserved OCSs ( i.e ., 70% viability compared to fresh-OCSs), as previously reported [21]. In this study, cryopreservation was carried out for only a short period (one week); further experiments are required to evaluate the effects of longer term cryopreservation.…”

“…Injected stem cells can resettle to a specific tissue owing to their homing characteristics [3] However, cryopreservation has been reported to have a negative effect on homing characteristics of stem cells [19], reducing the homing ability of stem cells. However, cell sheets can be used in a localized area, therefore, cell sheets can be used more efficiently than stem cells [21]. Moreover, gelatin contains an arginine-glycine-aspartic acid (RGD) sequence, which promotes cells stability with ECM [40] and cell adhesion through integrin [4142].…”

“…Frozen-thawed GCSs (FT-GCSs) were prepared using 3rd passage GCSs and the previously described slow-freezing method [21]. After 10 days of differentiation in gelatin-containing differentiation medium, cell sheets were collected with the help of a cell scraper and transferred to 2 mL cryovials (cryogenic vial; BD Falcon).…”

“…Contrary to the application of stem cells, stem cell sheets can be applied regionally to provide an improved localized effect [101113]. Also, frozen-thawed OCSs can maintain their osteogenic potential and produce a highly mineralized matrix in bone defective sites [21].…”