Osteogenic Extracellular Matrix Sheet and Its Healing Potential of Allogenic Implantation in a Rat Femoral Nonunion Model

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Grant support received from: Japan Orthopaedics and Traumatology Research Foundation, Inc. and the Translational Research Network Program from the Japan Agency for Medical Research and Development, AMED There is no financial information to disclose. Minimally invasive percutaneous injection of osteogenic cell sheet (OCS) could increase bone regeneration in a rat nonunion model, but the allogenic implantation may have immunological rejection. We hypothesized that the osteogenic extracellular matrix sheet (OECMS), which has no cellular component, can retain growth factors integrity and enhance bone formation in a rat nonunion model. In an in vitro study, OCSs were fabricated from rat bone marrow stromal cells and OECMSs were devitalized from OCSs with liquid nitrogen. Bone morphogenetic protein 2 (BMP-2),vascular endothelial growth factors (VEGF)-A, and basic fibroblast growth factor (bFGF) concentrations in the OECMS were quantified using ELISA. In an in vivo study, we implanted OECMS in a rat femoral nonunion model. Eighteen male Fischer 344 inbred rats were divided into 2 groups, the OECMS group and the control group. Fracture healing was evaluated with radiological and histological analyses at 2, 5, and 8 weeks. Biomechanical analysis was performed at 8 weeks. Compared with the OCS group, cell viability of the OECMS group was 0.2%. BMP-2, VEGF-A, and bFGF was retained in the OECMS (BMP-2: OCS, 86 pg/ml; OECMS, 86 pg/ml, VEGF-A: OCS, 6,944 pg/ml; OECMS, 4,056 pg/ml, bFGF: OCS, 720 pg/ml; OECMS, 448 pg/ml) (Figure 74-1). Radiological evaluations demonstrated enhanced bone regeneration in the OECMS group compared with the control group at 8 weeks (union rate OECMS group: 57%, control group: 0%) . Histological evaluations demonstrated bridging callus over the fracture site in the OECMS group at 8 weeks (Figure 74-2). Biomechanical testing revealed a significantly higher maximum bending load and stiffness in the OECMS group compared with the control group. (OECMS, 45.2 ± 24 N; control, 10.3 ± 9.6 N; P < .05, OECMS, 116 ± 99 N/mm; control, 25.7 ± 21 N/mm; P < .05, respectively). •Cell viability of the OECMS group was significantly decreased compared with that of the OCS group.•OECMS contained rich BMP-2, VEGF-A, and basic FGF after the freeze-thaw cycling method using liquid nitrogen.•OECMS represents a novel sheet-shaped scaffold with bone healing potential and contains rich osteoinductive and osteoconductive ability.74-2Histological findings of fracture sites at 2, 5, and 8 weeks in osteogenic extracellular matrix sheet (OECMS) and control groups. At 2 weeks, large soft tissue covered the fracture site in the OECMS group; however, fibrous tissue was interpositioned between the ends of the fractured bone in the control group. At 5 weeks, the calcification was expanded onto the periosteum in the OECMS group. However, calcification was not confirmed over the fracture site in the control group. At 8 weeks after surgery, bridging callus over the fracture site was observed in the OECMS group. However, the control group showed fibrous granulation tissue at the fracture site, the ends of cortical bone had become rounded, and there was evidence of bony resorption.View Large Image Figure ViewerDownload Hi-res image Download (PPT)