摘要：

Introduction: Enhancement of bone regeneration is needed to heal large bone defects and to treat fracture-delayed unions ornonunions. In addition, osteoporotic fractures are common in the elderly population. Therefore, developing an effectivetechnology for large bone repair is critical in improving the nation's health. Rapid advances in regenerative medicine have raised the hope of repairing bone defects with the combination of biomaterials, cells and growth factors [1]. A major challenge in cell- based bone tissue engineering is to promote efficient osteogenic differentiation of cells seeded within three-dimensional (3D)biomaterial scaffolds, which requires the sustained introduction of soluble osteoinductive biofactors [2], such as bonemorphogenetic protein-2 (BMP-2). However, their short half-lives and rapid clearance by the bloodstream as seen inexperimental animals limit the ability of these biofactors. Recombinant adeno-associated viral (rAAV) vector-based geneticengineering of stem cells is a promising therapeutic approach to promote bone tissue healing [3]. However, traditional ex vivogene transfer of BMP-2 to stem cells, prior to their seeding onto biomaterial scaffolds for tissue engineering and implantation, is a time consuming process and requires complicated cell culture in vitro [4]. The current study builds on the recent technological advancements in our labs in the design and fabrication of a novel 3D porous scaffold for bone tissue engineering. The ice-based microparticle porogenization method [5] enables the single-step fabrication of regenerative bone scaffolds encapsulated withhuman bone marrow mesenchymal stem cells (hBMSCs) and viral vector encoding BMP-2. We report here the efficientexpression of BMP-2 in vitro and osteoinductive effects on naive hBMSCs implanted in the hindlimb muscle of SCID mice.Methods: Both human BMP-2 and the green fluorescent protein (GFP) reporter genes under control of the CMV promoter were constructed in a self-complementary AAV vector (scAAV), respectively. Serotypes 2, 6 and 8 of the scAAV-CMV-GFP vectors were produced according to our published protocol [6]. hBMSCs were isolated with IRB approval from the femoral heads of patientsundergoing total hip arthroplasty. Poly-L-lactide (Sigma, Lot#SLBD6608V) was dissolved in chloroform (10 wt %) and cooled to - 20 ° C overnight. Ice-based microparticles were generated by injecting AAV solution diluted in deionized water throughspecialized nozzles (World Precision Instruments, Inc., Cat: TIPO5TW1F-L) into liquid nitrogen (Figure 1A). PLLA solution and the AAV ice micro-particles were mixed evenly in pre-cold stainless steel container and shaped by pre-cold stainless steel molds ondry ice. Finally, the 4x5x5 mm³ scaffold samples were kept in liquid nitrogen overnight and freeze-dried in lyophilizer for 4 hours. For structural observation, samples without AAV were bisected just before freeze-drying. After fabrication, scaffolds weresterilized by 70% ethanol for 10 minutes and rinsed once with phosphate-buffered saline (PBS). 20 ul (about 1.6x10^5 cell) ofhBMSC cell suspension were then seeded evenly into the AAV-activated PLLA scaffolds. Cell viability was assessed via Calceinacetoxymethyl ester (calcein-AM) and ethidium homodimer-1 (EthD-1) staining (Live/Dead Kit, Invitrogen) after 24 hrs and 7days. After the entire fabrication process, the activity of the AAV vector was also tested by release kinetics analysis. BMP-2concentration in the medium after 7 days was measured by BMP-2 ELISA kit (R&D). To test the efficacy of the AAV6-CMV-BMP-2 activated porous scaffold in enhancing bone formation in vivo, we implanted the hBMSCs-seeded BMP-2 gene-activated porous scaffolds within the hindlimb muscle in immune-deficient (SCID) mice. Bone formation was vitally monitored with micro-CT using Scanco VivaCT40 system.Results: 1. Fabriaction of AAV gene-activated PLLA porous scaffold: Ice-based micro-particles were generated with highefficiency with a median diameter of 250 µm, ranging from 100 to 500 µm, as shown in Figure 1B. As shown

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