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.
Highlights
With rapid advancements in tissue engineering, various skeletal diseases and complications—such as osteonecrosis and nonunion—can be treated using tissue-engineered bone derived from bone marrow-derived mesenchymal stem cells (BMSCs) [1]-[4]
To reduce the cell preparation time required prior to transplantation, we developed a method of osteogenic matrix cell sheets (OMCSs) cryopreservation and transplantation that can be used for skeletal reconstruction
Osteogenesis of Injected Cryopreserved OMCSs at Ectopic Sites The macroscopic appearances of harvested specimens obtained from the fresh, rapid, slow-freezing groups are shown in Figure 3(A), Figure 3(G) and Figure 3(M)
Summary
With rapid advancements in tissue engineering, various skeletal diseases and complications—such as osteonecrosis and nonunion—can be treated using tissue-engineered bone derived from bone marrow-derived mesenchymal stem cells (BMSCs) [1]-[4]. A cell sheet transplantation technique for bone regenerative medicine was recently developed using BMSCs [14]-[16]. OMCSs show high osteogenic potential in vitro and in vivo after subcutaneous scaffold-free transplantation [14] and in combination with artificial bone, such as beta-tricalcium phosphate [16]. Experimental animal models have successfully demonstrated the ability for OMCSs to treat fracture nonunion [15] and ligament reconstruction [17] by enhancing bone union and callus formation between bones, as well as ligamentous surfaces and bone tunnels. Tissue invasion by injectable bone may be small, as transplantation is conducted only by injection; this method can be used to treat delayed union and fracture nonunion with repeated cell sheet transplantation
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