Three-dimensional printed beta-TCP scaffold for bone tissue engineering

Statement of the Problem: The use of autologous tissue-engineered bone would eliminate the problem of donor site morbidity. Successful tissue engineered bone grafts will be dependent on the fabrication of accurate three-dimensional scaffolds. Recently, three-dimensional printing technology (3DPT) developed at the Massachusetts Institute of Technology has allowed the fabrication of geometry-controlled scaffolds. The purpose of this study is to evaluate cell penetration and bone formation on such a 3DPT scaffold. Materials and Methods: Beta-tricalcium phosphate (beta-TCP) scaffolds (Experimental Scaffolds) were made in as a 3D block measuring 20 20 7 mm in size. These scaffolds were fabricated using a TheriForm machine (Series 3200; Therics Inc, Princeton, NJ) and had both large (2 mm in diameter) and small pores (400 microns in diameter). Control scaffolds were made manually using poly-DL-lactic-co-glycolic acid (PLGA). PLGA foam squares cut into chips measuring 7 7 2 mm each. The PLGA chips were placed in a 20 20 7 mm mold and placed in an oven for 8 hours at 65° centigrade. The heat caused the PLGA to melt at the surface, binding the individual foam chip squares together, forming a single, three-dimensional polymer scaffold with an interconnected pore structure (“fused interconnected scaffold”). Porcine mesenchymal stem cells (pMSCs) were isolated from minipig (age 6 months; n 3) iliac crest marrow. The pMSCs were suspended in medium containing osteogenic supplements (OS: 100 nM dexamethasone, 50 g/mL ascorbic acid, and 10 mM -glycerophosphate) and then diluted to 30 106 cells per 35 mL OS medium. The cell suspension was seeded onto PLGA solid and fused interconnected scaffolds. The seeded scaffolds were placed in a rotational oxygenpermeable bioreactor system (ROBS) and incubated for 2, 4, and 6 weeks. Method of Data Analysis: After incubation, scaffolds were examined histologically to assess cell penetration and bone formation. The number of cells in the center of the scaffold was counted in 3 areas and averaged (n 3) using image analysis software (NIH Image 1.62 graphics program; NIH, Bethesda, MD). Bone formation depth was assessed in 3 areas of the scaffold cultured for 6 weeks and averaged (n 3) (Meta Morph version 6.6v4, Universal Imaging Corporation, Downingtown, PA). Results: At 2, 4, and 6 weeks, beta-TCP scaffold exhibited cell penetration of 21, 16.7, 19.6 cells/1.4 mm2 as compared to the fused interconnected scaffold (116.9, 74.3, and 60.4 cells/1.4 mm2). However, beta-TCP scaffolds exhibited comparable bone formation (0.48 mm) to the fused interconnected scaffold (0.42 mm). Conclusion: Beta-TCP scaffold showed less cell penetration but comparable bone formation as compared to the fused interconnected scaffolds in this in vitro model.