In the treatment of osseous defects micro-structured three-dimensional materials for bone replacement serve as leading structure for cell migration, proliferation and bone formation. The scaffold design and culture conditions are crucial for the limited diffusion distance of nutrients and oxygen. In static culture, decreased cell activity and irregular distribution occur within the scaffold. Dynamic conditions entail physical stimulation and constant medium perfusion imitating physiological nutrient supply and metabolite disposal. Therefore, we investigated the influence of different scaffold configurations and cultivation methods on human osteoblasts. Cells were seeded on three-dimensional porous Ti-6Al-4V scaffolds manufactured with selective laser melting (SLM) or electron beam melting (EBM) varying in porosity, pore size and basic structure (cubic, diagonal, pyramidal) and cultured under static and dynamic conditions. Cell viability, migration and matrix production were examined via mitochondrial activity assay, fluorescence staining and ELISA. All scaffolds showed an increasing cell activity and matrix production under static conditions over time. Expectations about the dynamic culture were only partially fulfilled, since it enabled proliferation alike the static one and enhanced cell migration. Overall, the SLM manufactured scaffold with the highest porosity, small pore size and pyramidal basic structure proved to be the most suitable structure for cell proliferation and migration.

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Abstract:The use of additive manufacturing technologies to produce lightweight or functional structures is widespread. Especially Ti 6 Al 4 V plays an important role in this development field and parts are manufactured and analyzed with the aim to characterize the mechanical properties of open-porous structures and to generate scaffolds with properties specific to their intended application. An SLM and an EBM process were used respectively to fabricate the Ti 6 Al 4 V single struts. For mechanical characterization, uniaxial compression tests and hardness measurements were conducted. Furthermore, the struts were manufactured in different orientations for the determination of the mechanical properties. Roughness measurements and a microscopic characterization of the struts were also carried out. Some parts were characterized following heat treatment (hot isostatic pressing). A functional correlation was found between the compressive strength and the slenderness ratio (λ) as well as the equivalent diameter (d) and the height (L) of EBM and SLM parts. Hardness investigations revealed considerable differences related to the microstructure. An influence of heat treatment as well as of orientation could be determined. In this work, we demonstrate the influence of the fabrication quality of single struts, the roughness and the microstructure on mechanical properties as a function of orientation.

Studies on bone cell ingrowth into synthetic, porous three-dimensional (3D) implants showed difficulties arising from impaired cellular proliferation and differentiation in the core region of these scaffolds with increasing scaffold volume in vitro. Therefore, we developed an in vitro perfusion cell culture module, which allows the analysis of cells in the interior of scaffolds under different medium flow rates. For each flow rate the cell viability was measured and compared with results from computer simulations that predict the local oxygen supply and shear stress inside the scaffold based on the finite element method. We found that the local cell viability correlates with the local oxygen concentration and the local shear stress. On the one hand the oxygen supply of the cells in the core becomes optimal with a higher perfusion flow. On the other hand shear stress caused by high flow rates impedes cell vitality, especially at the surface of the scaffold. Our results demonstrate that both parameters must be considered to derive an optimal nutrient flow rate.

Bone loss in the near-vicinity of implants can be a consequence of stress shielding due to stiffness mismatch. This can be avoided by reducing implant stiffness, i.e., by implementing an open-porous structure. Three open-porous designs were therefore investigated (cubic, pyramidal and a twisted design). Scaffolds were fabricated by a selective laser-melting (SLM) process and material properties were determined by conducting uniaxial compression testing. The calculated elastic modulus values for the scaffolds varied between 3.4 and 26.3 GP and the scaffold porosities between 43% and 80%. A proportional linear correlation was found between the elastic modulus and the geometrical parameters, between the elastic modulus and the compressive strengths, as well as between the strut width-to-diameter ratio (a/d) and elastic modulus. Furthermore, we found a power-law relationship between porosity and the modulus of elasticity that characterizes specific yielding. With respect to scaffold porosity, the description of specific yielding behaviour offers a simple way to characterize the mechanical properties of open-porous structures and helps generate scaffolds with properties specific to their intended application. A direct comparison with human bone parameters is also possible. We generated scaffolds with mechanical properties sufficiently close to that of human cortical bone.

Additive manufacturing of lightweight or functional structures by selective laser beam (SLM) or electron beam melting (EBM) is widespread, especially in the field of medical applications. SLM and EBM processes were applied to prepare Ti6Al4V test specimens with different surface orientations (0°, 45° and 90°). Roughness measurements of the surfaces were conducted and cell behavior on these surfaces was analyzed. Hence, human osteoblasts were seeded on test specimens to determine cell viability (metabolic activity, live-dead staining) and gene expression of collagen type 1 (Col1A1), matrix metalloprotease (MMP) 1 and its natural inhibitor, TIMP1, after 3 and 7 days. The surface orientation of specimens during the manufacturing process significantly influenced the roughness. Surface roughness showed significant impact on cellular viability, whereas differences between the time points day 3 and 7 were not found. Collagen type 1 mRNA synthesis rates in human osteoblasts were enhanced with increasing roughness. Both manufacturing techniques further influenced the induction of bone formation process in the cell culture. Moreover, the relationship between osteoblastic collagen type 1 mRNA synthesis rates and specimen orientation during the building process could be characterized by functional formulas. These findings are useful in the designing of biomedical applications and medical devices.

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