Cell Sheet Injection as a Technique of Osteogenic Supply

Ueha²,

Shimizu³

et al. 2012

SCD

We have previously reported on both the osteogenic potential of hydroxyapatite (HA) combined with bone marrow-derived mesenchymal stem cells (BMSCs) and a method involving osteogenic matrix cell sheet transplantation of BMSCs. In the present study, we assessed the osteogenic potential of serially-passaged BMSCs, both in vitro and in vivo. We also assessed whether an additional cell-loading technique can regain the osteogenic potential of the constructs combined with serially-passaged BMSCs. The present study revealed that passage (P) 1 cells cultured in osteogenic-induced medium showed strong positive staining for alkaline phosphatase (ALP) and Alizarin Red S, whereas P3 cells showed faint staining for ALP, with no Alizarin Red S staining. Staining of P1, P2 and P3 cells were progressively weaker, indicating that the osteogenic potential of the serially-passaged rat BMSCs is lost after P3 in vitro. The in vivo study showed that little bone formation was observed in the HA constructs seeded with P3 cells, 4 weeks after subcutaneous implantation. However, P3 cell/HA constructs which had increased cell-loading showed abundant bone formation within the pores of the HA construct. ALP and osteocalcin mRNA expression in these constructs was significantly higher than that of constructs with regular cell-seeding. The present study indicates that the osteogenic potential of the constructs with serially-passaged BMSCs is increased by additional cell-loading. This method can be applied to cases requiring hard tissue reconstruction, where BMSCs require serial expansion of cells.

Section: Rna Isolation and Real-time Quantitative Pcrmentioning

confidence: 99%

Increased osteogenesis with hydroxyapatite constructs combined with serially-passaged bone marrow-derived mesenchymal stem cells

Ueha²,

Shimizu³

et al. 2012

SCD

“…To measure the messenger (m)RNA expression levels, real-time quantitative PCR (ABI StepOne Plus Real Time PCR System), using primers for rat cDNAs, as previously described. 24 The target mRNA levels were compared after normalising to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels as an internal standard that corrected for differences in the efficiency of reverse transcription between samples. The primers for the target mRNAs were collagen type I (Rn00801649 g1), laminin (Rn00564264 m1), fibronectin (Rn00569575 m1), osteocalcin (Rn01455285 g1) and GAPDH (Rn99999916 s1).…”

Section: Methodsmentioning

confidence: 99%

“…22-24 We have previously proposed solutions to undertake difficult tissue regeneration, including fracture nonunion 25 and ligament reconstruction 26 using scaffold-free cell sheet transplantation. We have also reported a technique for cell sheet injection we call ‘injectable bone’, that promotes osteogenesis in necrotic bone and implanted materials.…”

Section: Introductionmentioning

confidence: 99%

“…We have also reported a technique for cell sheet injection we call ‘injectable bone’, that promotes osteogenesis in necrotic bone and implanted materials. 24,27 The method we previously reported to create osteogenic matrix cell sheets from BMSCs requires culturing the cells with both Dex and AscP to create confluent cells in the structure of sheets that can be mechanically retrieved. 22,27 In our previous reports, however, we did not determine whether culture of the cells with Dex or AscP alone induces sufficient structure to allow for the mechanical retrieval of the confluent cells in the structure of sheets.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Culturing bone marrow cells with dexamethasone and ascorbic acid improves osteogenic cell sheet structure

Shimizu

Kira

et al. 2016

Bone & Joint Research

Self Cite

ObjectivesTo assess the structure and extracellular matrix molecule expression of osteogenic cell sheets created via culture in medium with both dexamethasone (Dex) and ascorbic acid phosphate (AscP) compared either Dex or AscP alone.MethodsOsteogenic cell sheets were prepared by culturing rat bone marrow stromal cells in a minimal essential medium (MEM), MEM with AscP, MEM with Dex, and MEM with Dex and AscP (Dex/AscP). The cell number and messenger (m)RNA expression were assessed in vitro, and the appearance of the cell sheets was observed after mechanical retrieval using a scraper. β-tricalcium phosphate (β-TCP) was then wrapped with the cell sheets from the four different groups and subcutaneously implanted into rats.ResultsAfter mechanical retrieval, the osteogenic cell sheets from the MEM, MEM with AscP, and MEM with Dex groups appeared to be fragmented or incomplete structures. The cell sheets cultured with Dex/AscP remained intact after mechanical retrieval, without any identifiable tears. Culture with Dex/AscP increased the mRNA and protein expression of extracellular matrix proteins and cell number compared with those of the other three groups. More bridging bone formation was observed after transplantation of the β-TCP scaffold wrapped with cell sheets cultured with Dex/AscP, than in the other groups.ConclusionsThese results suggest that culture with Dex/AscP improves the mechanical integrity of the osteogenic cell sheets, allowing retrieval of the confluent cells in a single cell sheet structure. This method may be beneficial when applied in cases of difficult tissue reconstruction, such as nonunion, bone defects, and osteonecrosis.Cite this article: M. Akahane, T. Shimizu, T. Kira, T. Onishi, Y. Uchihara, T. Imamura, Y. Tanaka. Culturing bone marrow cells with dexamethasone and ascorbic acid improves osteogenic cell sheet structure. Bone Joint Res 2016;5:569–576. DOI: 10.1302/2046-3758.511.BJR-2016-0013.R1.

“…Cell sheets have been created from the skin [7], myocardial cells [8] [9], periodontal ligaments [10], corneal epithelia [11], bladder epithelia [12], and mucosal epithelia [13]. A cell sheet transplantation technique for bone regenerative medicine was recently developed using BMSCs [14]- [16]. BMSCs are cultured in media containing dexamethasone (Dex) and ascorbic acid phosphate (AscP), and then lifted as single cell sheets using a scraper.…”

Section: Introductionmentioning

confidence: 99%

Use of Cryopreserved Osteogenic Matrix Cell Sheets for Bone Reconstruction

Kura

Shimizu

et al. 2016

SCD

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.