One-Step Fabrication of Bone Morphogenetic Protein-2 Gene-Activated Porous Poly-L- Lactide Scaffold for Bone Induction  Jingwen Xue, Hang Lin, Allison Bean, Ying Tang, Jian Tan, Rocky S. Tuan, Bing Wang  Molecular Therapy - Methods & Clinical Development  Volume 7, Pages 50-59 (December 2017) DOI: 10.1016/j.omtm.2017.08.008 Copyright © 2017 The Authors Terms and Conditions

Figure 1 Schematic Representation of the Preparation of AAV-Activated PLLA Scaffold (A) Concentrated AAV is diluted in PBS and injected through specialized nozzles into liquid nitrogen. Ice microparticles containing AAV are formed, with diameters ranging from 100 to 500 μm. (B) The AAV-containing microparticles are mixed with PLLA solubilized in chloroform (pre-chilled to −20°C). The scaffold is fabricated and porogenized with ice-based microparticles. (C) The porous AAV-PLLA scaffold is formed upon removal of solvent and water by lyophilization, with the ice microparticles acting as the micro-porogen. Enlarged views are provided to represent dispersal of AAV particles onto the walls of the pores within the scaffold upon lyophilization. (D) hBMSCs are seeded into the porous AAV-PLLA scaffold and are subsequently exposed to and infected by the AAV particles released from the interior wall of the pores within the scaffolds, as depicted in the enlarged view of the pores. Molecular Therapy - Methods & Clinical Development 2017 7, 50-59DOI: (10.1016/j.omtm.2017.08.008) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Characterization of Ice-Porogenized PLLA Porous Scaffold (A and B) Ice microparticles used as a porogen (A), with diameters ranging from 100 to 500 μm (B). Scale bar, 100 μm. (C) SEM image of cross-section of porous PLLA scaffold fabricated using ice-based microparticles (circle indicates pore, with a diameter of around 300–400 μm). Scale bar, 100 μm. (D) Macroscopic view of PLLA porous scaffold fabricated with ice-based microparticles. Molecular Therapy - Methods & Clinical Development 2017 7, 50-59DOI: (10.1016/j.omtm.2017.08.008) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Cell Viability of hBMSCs Laden on AAV-Activated Porous PLLA Scaffolds (A) Live/dead staining of cells seeded within AAV-activated scaffold surface 1 day (left) and 7 days (right) after seeding. Scale bar, 100 μm. (B) CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) analysis of metabolic activity of hBMSCs seeded in PLLA scaffolds fabricated with or without AAV-BMP2 viral constructs and on two-dimensional plates at culture days 1 and 7. All data were normalized to data from hBMSCs cultures on plain PLLA scaffolds (fabricated without AAV-BMP2 constructs) on day 1. Molecular Therapy - Methods & Clinical Development 2017 7, 50-59DOI: (10.1016/j.omtm.2017.08.008) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Release Kinetics of AAV Particles from Gene-Activated Porous PLLA Scaffolds (A) Dot blot quantification of daily release of AAV-GFP v.g. of particles in incubation medium up to day 20 (initial load = 5 × 1011 v.g. of particles per scaffold). (B) The cumulative release over time clearly indicated fast and major release from day 1 to 10 and subsequent slow and minor release. n = 3. Molecular Therapy - Methods & Clinical Development 2017 7, 50-59DOI: (10.1016/j.omtm.2017.08.008) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Effect of Various 2D and 3D Culture Conditions on BMP2 and Osteogenic Gene Expression by hBMSCs after 2 Weeks of Culture (A) BMP2 level (pg/mL) determined by ELISA. (B and C) Relative gene expression of OCN (B) and BSP2 (C) by qRT-PCR. Data for fold change of gene expression are normalized to those of 2D hBMSC cultures without BMP2 infection. Molecular Therapy - Methods & Clinical Development 2017 7, 50-59DOI: (10.1016/j.omtm.2017.08.008) Copyright © 2017 The Authors Terms and Conditions

Figure 6 Bone Formation In Vivo after Implantation of hBMSC-Seeded AAV-BMP2 Gene-Activated Constructs Four groups of hBMSC-seeded PLLA scaffolds were used: (1) AAV-BMP2 activated; (2) AAV-GFP activated; (3) laden with BMP2 protein; and (4) control. (A) Micro-CT detection of ectopic bone formation at 4 weeks post-implantation. Bone formation was detected in the implantation site (circles) of an AAV-BMP2-activated scaffold seeded with hBMSCs, but not in similar areas with an AAV-GFP-activated or BMP2 protein laden scaffold or in a scaffold seeded only with hBMSCs. (B and C) Three-dimensional micro-CT reconstruction (B) and mean density (C) of ectopic neo-bone tissue formation in hBMSC-seeded AAV gene-activated PLLA constructs as a function of time after implantation. Molecular Therapy - Methods & Clinical Development 2017 7, 50-59DOI: (10.1016/j.omtm.2017.08.008) Copyright © 2017 The Authors Terms and Conditions

Figure 7 Quantification of Calcium Accumulation in hBMSC-Seeded PLLA Scaffold-Based Implants at 10 Weeks Post-implantation Three groups of PLLA scaffolds were used: (1) AAV-BMP2 activated; (2) laden with BMP2 protein; and (3) control. Gene-activated scaffold-based constructs showed a statistically higher level of calcium accumulation. Molecular Therapy - Methods & Clinical Development 2017 7, 50-59DOI: (10.1016/j.omtm.2017.08.008) Copyright © 2017 The Authors Terms and Conditions

Figure 8 Histological Analysis of hBMSC-Seeded PLLA Scaffold-Based Implants at 10 Weeks Post-implantation by H&E (Top Row) and Herovici Staining (Bottom Row) Three groups of PLLA scaffolds were used: (1) AAV-BMP2 activated; (2) laden with BMP2 protein; and (3) control. Robust ectopic neo-bone formation is seen in the AAV-BMP2 gene-activated scaffold group. Scale bar, 100 μm. Molecular Therapy - Methods & Clinical Development 2017 7, 50-59DOI: (10.1016/j.omtm.2017.08.008) Copyright © 2017 The Authors Terms and Conditions