Bone Regeneration by Mesenchymal Stem Cell Sheets Overexpressing BMP-7 in Canine Bone Defects

Abstract

Successful repair of bone defect injuries is a major issue in reconstructive surgery. The necessary elements for bone healing include osteogenic cells, osteoinductive growth factors, and osteoconductive matrix. Multipotent mesenchymal stem cells (MSCs) and MSC sheets have potential for bone regeneration. Bone morphogenetic protein 7 (BMP-7) has been shown to possess strong osteoinductive properties. In addition, composite polymer/ceramic scaffolds such as poly ε-caprolactone (PCL)/β-tricalcium phosphate (β-TCP) are widely used to repair large bone defects. In the first chapter, the in vitro osteogenic potential of canine adipose-derived MSCs (Ad-MSCs) and Ad-MSC sheets are compared. Composite PCL/β-TCP scaffolds seeded with Ad-MSCs or wrapped with osteogenic Ad-MSC sheets (OCS) were also fabricated and their in vivo osteogenic potential was assessed after transplantation into critical-sized bone defects in dogs. The alkaline phosphatase (ALP) activity in osteogenic Ad-MSCs (O-MSCs) and OCS was significantly higher than that of undifferentiated Ad-MSCs (U-MSCs). ALP, runt related transcription factor 2 (RUNX2), osteopontin, and BMP-7 mRNA levels were up-regulated in O-MSCs and OCS compared to those in U-MSCs. The amount of newly formed bone was greater in PCL/β-TCP/OCS and PCL/β-TCP/O-MSCs/OCS than in the PCL/β-TCP/U-MSCs and PCL/β-TCP/O-MSCs groups. Although there was no difference between in vitro osteogenic genes expression of O-MSCs and OCS, the new bone formation was greater in the scaffold wrapped with OCS than the scaffold seeded U-MSCs and O-MSCs. Consequently, it was suggested that OCS could be used as an osteogenic matrix in canine critical-sized bone defects. OCS is difficult to handle and culture for more than two weeks

however, undifferentiated Ad-MSC sheets (UCS) are easy to culture and handle. The osteogenic capacity of USCs could be enhanced by canine BMP-7 gene transduction using a lentiviral system. Demineralized bone matrix (DBM), as defect filling and osteoinductive materials in large bone defects, was combined in a subsequent study. Combination of UCS overexpressing BMP-7 and DBM is supposed to be potential vehicles for bone healing. In the second chapter, canine Ad-MSCs overexpressing BMP-7 (BMP-7-MSCs) were produced and sheets formed from these cells (BMP-7-CS) were compared with Ad-MSC sheets for in vitro osteogenic potential. BMP-7-CS with and without DBM, were transplanted into critical-sized bone defects in vivo and osteogenesis was assessed. BMP-7 mRNA and protein levels were up-regulated in BMP-7-MSCs compared to those in Ad-MSCs. The ALP activity in Ad-MSC sheets and BMP-7-CS were significantly higher than that in Ad-MSCs. ALP, RUNX2, osteopontin, BMP-7, transforming growth factor-β and platelet-derived growth factor-β mRNA levels were up-regulated in BMP-7-CS compared to levels in Ad-MSCs and Ad-MSC cell sheets. BMP-7-CS showed the highest osteogenic abilities in vitro. The amount of newly formed bone and neovascularization were greater in BMP-7-CS and BMP-7-CS/DBM groups than in control groups. However, the BMP-7-CS/DBM group had more mineralized particles inside the defect sites than the BMP-7-CS group. As a result, it was suggested that a combination of BMP-7-CS and DBM could be used as osteogenic materials in canine critical-sized bone defects. BMP-7-CS not only provides BMP-7 producing MSCs but also produce osteogenic and vascular trophic factors. The combination of these cells with the osteoinduction and osteoconduction properties of DBM could result in synergy during bone regeneration. Thus, transplantation of BMP-7-CS and DBM could be used as an alternative treatment modality in bone tissue engineering.