Flow Perfusion Improves Seeding of Tissue Engineering Scaffolds with Different Architectures

Abstract

Engineered bone grafts have been generated in static and dynamic systems by seeding and culturing osteoblastic cells on 3-D scaffolds. Seeding determines initial cellularity and cell spatial distribution throughout the scaffold, and affects cell-matrix interactions. Static seeding often yields low seeding efficiencies and poor cell distributions; thus creating a need for techniques that can improve these parameters. We have evaluated the effect of oscillating flow perfusion on seeding efficiency and spatial distribution of MC3T3-E1 pre-osteoblastic cells in fibrous polystyrene matrices (20, 35 and 50-microm fibers) and foams prepared by salt leaching, using as controls statically seeded scaffolds. An additional control was investigated where static seeding was followed by unidirectional perfusion. Oscillating perfusion resulted in the most efficient technique by yielding higher seeding efficiencies, more homogeneous distribution and stronger cell-matrix interactions. Cell surface density increased with inoculation cell number and then reached a maximum, but significant detachment occurred at greater flow rates. Oxygen plasma treatment of the fibers greatly improved seeding efficiency. Having similar porosity and dimensions, fibrous matrices yielded higher cell surface densities than foams. Fluorescence microscopy and histological analyses in polystyrene and PLLA scaffolds demonstrated that perfusion seeding produced more homogeneous cell distribution, with fibrous matrices presenting greater uniformity than the foams.

Effect of surface acoustic waves on the viability, proliferation and differentiation of primary osteoblast-like cells

“…As compared to the methods described in the literature, 4,[6][7][8][9][10][11][12] where the seeding process usually takes hours to days, the present SAW method significantly accelerates the cell seeding process. The fast seeding time also means that multiple drops containing the cells can be successively driven into the scaffold to achieve greater cell loading.…”

“…As we mentioned before, there are various cell seeding methods being developed and most of these studies investigate the cell viability and functionality. 4,[6][7][8][9][10][11][12] Undoubtedly, cell viability and functionality are critical to the usefulness of any new technique for tissue engineering. 5 Therefore, in addition to high cell viability, the proliferation and differentiation ability of the treated cells showed no difference from those of the untreated cells, indicating that the SAW has no deleterious effects on the cells' functionality.…”

“…However, low seeding efficiencies and nonuniform cell distributions have been reported with these methods as well. [3][4][5][6][7][8][9][10][11][12] Alvarez-Barreto and co-workers 10,11 developed a more refined approach from these ideas using flow perfusion; a cell suspension is first poured on top of the prewetted scaffold and then the flow is drawn into the scaffold by oscillatory pumping across it, driving the fluid with the suspended cells into the scaffold matrix. Compared to past methods, this technique has been reported to deliver superior cell adhesion and seeding efficiency.…”

“…However, even this perfusion method is slow, requiring 1-2 h. Moreover, large fluid pumps are required to compensate for the large pressure drops associated with the high capillary stresses resisting the flow of the fluid through the scaffold, a notable limitation in miniaturizing the technology to dimensions appropriate for point-of-care use. 10,11 We reported in a previous work another method 13 using surface acoustic wave ͑SAW͒ radiation 14 to provide the perfusion force. In that work, we demonstrated that small ͑10 ᐉ͒ suspensions of microparticles may be rapidly driven into a porous polycaprolactone ͑PCL͒ scaffold in 10 s, some three to four orders of magnitude faster than the hour-long process required using the static and perfusion methods described above.…”

Simulation of Cell Seeding Within a Three-Dimensional Porous Scaffold: A Fluid-Particle Analysis

“…Studies on seeding efficiency by static deposition have reported results ranging from 18% to 85%. 3,4,6,[10][11][12] Static seeding also presents the disadvantage of leading to a nonuniform cell distribution. 3,4,10 Dynamic cell seeding with bioreactors has proven to provide a higher efficiency and more even distribution of cells, 13 more particularly within 3D scaffolds, where perfusion systems were reported to lead to greater efficiency and better cell distribution.…”