Osteogenesis of osteogenic matrix cell sheets preserved in culture medium in a rat model

Osteogenic matrix cell sheets (OMCSs) are ideal for bone regeneration. Transportation of OMCSs may be necessary, during which their osteogenic ability must be maintained. Here, we evaluated different media and temperatures for OMCS preservation. Bone marrow stromal/stem cells (BMSCs) were obtained from Fischer rats and analyzed for stem cell markers by flow cytometry. OMCSs were prepared from BMSCs by treatment with dexamethasone and ascorbic acid phosphate. After OMCS collection, they were stored in minimum essential medium (MEM) or Hank's balanced salt solution (HBSS) at 37, 22, or 4°C for 24 hours. Cell viability and cytotoxic effects in the preservation conditions were determined by adenosine triphosphate (ATP) contents and lactate dehydrogenase (LDH) release, respectively. Osteogenesis was assessed by subcutaneously implanting preserved OMCSs around β-tricalcium phosphate ceramic disks into syngeneic rats. Implants were evaluated by alkaline phosphatase (ALP) activities, osteocalcin contents, and histology. Mesenchymal stem cells comprised 51% of primary cultured BMSCs. ATP contents were significantly different in OMCSs stored in MEM or HBSS at 22°C and 4°C. LDH release was significantly different in OMCSs stored in HBSS at 22°C and 4°C. The highest LDH release was observed in OMCSs stored in HBSS at 37°C. ALP activities and osteocalcin contents were the lowest in implanted OMCSs stored in HBSS at 37°C at four weeks after subcutaneous implantation. There was a significant difference in the osteocalcin levels of implanted OMCSs stored in MEM at 37°C and HBSS at 4°C. Abundant bone tissue around and inside disks was found in histological sections of OMCSs stored in all preservation conditions except for MEM and HBSS at 37°C. Maintaining the osteogenic ability of OMCSs during transport is important, and preservation of OMCSs in MEM or HBSS at 4°C or 22°C is a simple and inexpensive method.