Tomoyuki Ueha

Scaffold-free cell sheet injection results in bone formation.

We previously reported a new cell transplantation method in which mesenchymal stem cells (MSCs) were cultured as cell sheets. The cultured MSC sheets showed high alkaline phosphatase (ALP) activities and osteocalcin (OC) contents. In the present study, we transplanted such sheets by injection to assess whether the injectable MSC sheets could form bone tissue at subcutaneous sites. At 4 weeks after the subcutaneous injection, the injected areas showed hard mass formation. Each mass consisted of newly formed bone, as evaluated by radiographic, histological and gene expression analyses as well as three‐dimensional computed tomography (3D‐CT). Histological analyses revealed extracellular bone matrix together with osteocytes and active osteoblasts. Real‐time PCR analyses showed high ALP and OC mRNA expressions. We also injected the cell sheets into dead bone to determine whether the lost osteogenic potential could be rescued, and histological analyses revealed that the injected cell sheets supplied osteogenic potential to the dead bone. The present study clearly indicates that osteogenic MSC sheets can be transplanted via injection through a needle and that bone formation results in the injected areas. Owing to its usage of a needle for fabrication of in vivo bone tissue, this injection method can be applied as a minimally invasive approach for hard tissue reconstruction. Copyright

Osteogenic Matrix Cell Sheet Transplantation Enhances Early Tendon Graft to Bone Tunnel Healing in Rabbits

The objective of this study was to determine whether osteogenic matrix cell sheets (OMCS) could induce bone formation around grafted tendons, thereby enhancing early stage tendon to bone tunnel healing in skeletally mature male Japanese white rabbits. First, the osteogenic potential of rabbit OMCS was evaluated. Then, the OMCS were transplanted into the interface between the grafted tendon and the bone tunnel created at the tibia. Histological assessments and biomechanical tensile testing were performed after 3 weeks. The rabbit OMCS showed high alkaline phosphatase (ALP) activity, positive staining of ALP, and osteogenic potential when transplanted subcutaneously with beta tricalcium phosphate disks. Newly formed bony walls and positive collagen type I staining were seen around the grafted tendon with OMCS transplantation, whereas such bony walls were thinner or less frequent without OMCS transplantation. Micro-computed tomography images showed significantly higher bone volume in the OMCS transplantation group. The pullout strength was significantly higher with OMCS (0.74 ± 0.23 N/mm2) than without OMCS (0.58 ± 0.15 N/mm2). These results show that OMCS enhance early tendon to bone tunnel healing. This method can be applied to cases requiring early tendon to bone tunnel healing after ligament reconstruction surgery.

Differences in Highly Cross-Linked Polyethylene Wear Between Zirconia and Cobalt-Chromium Femoral Heads in Japanese Patients: A prospective, randomized study

The purpose of this study was to compare highly cross-linked polyethylene wear between the zirconia head and the cobalt-chromium head in Japanese patients. A prospective, randomized study was performed to evaluate the outcomes in 32 hips that had zirconia heads and in 30 hips that had cobalt-chromium heads. The mean follow-up periods of both groups were same (5 years). There were no significant differences between the zirconia head and the cobalt-chromium head in the mean polyethylene linear wear per year and the mean volumetric polyethylene wear per year in the steady phase. This study indicates that zirconia head offers no benefits over metal head in terms of wear reduction at 5 years in Japanese patients who have lightweight and thin polyethylene liners.

Osteogenesis of cryopreserved osteogenic matrix cell sheets

Cryopreservation of tissue engineered bone (TEB), whilst maintaining its osteogenic ability, is imperative for large-scale clinical application. We previously reported a novel cell transplantation method, in which bone-marrow-derived mesenchymal stem cells (BMSCs) were cultured to confluence and differentiated down the osteogenic lineage to form osteogenic matrix cell sheets (OMCS). OMCS have high alkaline phosphatase (ALP) activity and osteocalcin (OC) contents and can be easily used for producing TEB. The aim of the present study was to investigate whether TEB produced by cryopreserved OMCS maintains sufficient osteogenic potential in vivo. OMCS were prepared and divided into three groups according to storage period of cryopreservation (fresh (no cryopreservation), 4 week and 12 week cryopreservation groups). OMCS were cryopreserved by storage in freezing medium (Cell Banker 1®) at -80 °C. Cryopreserved OMCSs were rapidly thawed at room temperature and wrapped around Hydroxyapatite (HA) scaffolds prior to implantation into subcutaneous sites in rats, to determine their in vivo bone-forming capability. The constructs were harvested 4 weeks after transplantation and examined histologically and biochemically. Histological analysis of the constructs showed extensive bone formation in the HA pores with high ALP activity and OC content detected in the cryopreservation groups. The present study clearly indicates that cryopreserved/thawed OMCS are still capable of producing mineralized matrix on scaffolds, resulting in bone formation. This cryopreservation technique could be applied for hard tissue reconstruction to ease the cell preparation method prior to time of use.

Utility of tricalcium phosphate and osteogenic matrix cell sheet constructs for bone defect reconstruction

AIM To determine the effects of transplanting osteogenic matrix cell sheets and beta-tricalcium phosphate (TCP) constructs on bone formation in bone defects. METHODS Osteogenic matrix cell sheets were prepared from bone marrow stromal cells (BMSCs), and a porous TCP ceramic was used as a scaffold. Three experimental groups were prepared, comprised of TCP scaffolds (1) seeded with BMSCs; (2) wrapped with osteogenic matrix cell sheets; or (3) both. Constructs were implanted into a femoral defect model in rats and bone growth was evaluated by radiography, histology, biochemistry, and mechanical testing after 8 wk. RESULTS In bone defects, constructs implanted with cell sheets showed callus formation with segmental or continuous bone formation at 8 wk, in contrast to TCP seeded with BMSCs, which resulted in bone non-union. Wrapping TCP constructs with osteogenic matrix cell sheets increased their osteogenic potential and resulting bone formation, compared with conventional bone tissue engineering TCP scaffolds seeded with BMSCs. The compressive stiffness (mean ± SD) values were 225.0 ± 95.7, 30.0 ± 11.5, and 26.3 ± 10.6 MPa for BMSC/TCP/Sheet constructs with continuous bone formation, BMSC/TCP/Sheet constructs with segmental bone formation, and BMSC/TCP constructs, respectively. The compressive stiffness of BMSC/TCP/Sheet constructs with continuous bone formation was significantly higher than those with segmental bone formation and BMSC/TCP constructs. CONCLUSION This technique is an improvement over current methods, such as TCP substitution, and is useful for hard tissue reconstruction and inducing earlier bone union in defects.

Osteogenic Matrix Cell Sheets Facilitate Osteogenesis in Irradiated Rat Bone

Reconstruction of large bone defects after resection of malignant musculoskeletal tumors is a significant challenge in orthopedic surgery. Extracorporeal autogenous irradiated bone grafting is a treatment option for bone reconstruction. However, nonunion often occurs because the osteogenic capacity is lost by irradiation. In the present study, we established an autogenous irradiated bone graft model in the rat femur to assess whether osteogenic matrix cell sheets improve osteogenesis of the irradiated bone. Osteogenic matrix cell sheets were prepared from bone marrow-derived stromal cells and co-transplanted with irradiated bone. X-ray images at 4 weeks after transplantation showed bridging callus formation around the irradiated bone. Micro-computed tomography images at 12 weeks postoperatively showed abundant callus formation in the whole circumference of the irradiated bone. Histology showed bone union between the irradiated bone and host femur. Mechanical testing showed that the failure force at the irradiated bone site was significantly higher than in the control group. Our study indicates that osteogenic matrix cell sheet transplantation might be a powerful method to facilitate osteogenesis in irradiated bones, which may become a treatment option for reconstruction of bone defects after resection of malignant musculoskeletal tumors.

Limitations of Joint-Preserving Treatment for Osteonecrosis of the Femoral Head: Limitation of Free Vascularized Fibular Grafting

Fifty-six hips of 46 patients undergoing free vascularized fibular grafting for the treatment of osteonecrosis of the femoral head were investigated. The average age at surgery was 39 years, and the average follow-up period was 6 years. Associated etiological factors included a history of high-dose steroids for 27 hips, consumption of alcohol for 25, and idiopathy for 4 hips. The radiographic appearance, determined according to the staging system of the Japanese Investigation Committee, was stage 1 for 2 hips, stage 2 for 28, stage 3A for 15, stage 3B for 10, and stage 4 for 1 hip. The radiographic type of necrosis, determined according to the radiographic classification of the Japanese Investigation Committee, was type B for 4 hips, type C-1 for 20, and type C-2 for 32 hips. The clinical results of steroid-induced osteonecrosis were poorest among the etiologies. Twenty-four hips collapsed or progressed radiographically. There was a significant relationship between preoperative stage and radiographic progression. There was also a significant relationship between preoperative type and radiographic progression. Eleven hips were converted to total hip arthroplasty. In conclusion, the current results show that vascularised fibular grafting is a good procedure for the precollapse stages and a valuable alternative for patients with stage 3A.