Effects of initial seeding density and fluid perfusion rate on formation of tissue-engineered bone

Abstract

We describe a novel bioreactor system for tissue engineering of bone that enables cultivation of up to six tissue constructs simultaneously, with direct perfusion and imaging capability. The bioreactor was used to investigate the relative effects of initial seeding density and medium perfusion rate on the growth and osteogenic differentiation patterns of bone marrow-derived human mesenchymal stem cells (hMSCs) cultured on three-dimensional scaffolds. Fully decellularized bovine trabecular bone was used as a scaffold because it provided suitable "biomimetic" topography, biochemical composition, and mechanical properties for osteogenic differentiation of hMSCs. Trabecular bone plugs were completely denuded of cellular material using a serial treatment with hypotonic buffers and detergents, seeded with hMSCs, and cultured for 5 weeks. Increasing seeding density from 30 x 10(6) cells/mL to 60 x 10(6) cells/mL did not measurably influence the characteristics of tissue-engineered bone, in contrast to an increase in the perfusion rate from 100 microms(-1) to 400 microms(-1), which radically improved final cell numbers, cell distributions throughout the constructs, and the amounts of bone proteins and minerals. Taken together, these findings suggest that the rate of medium perfusion during cultivation has a significant effect on the characteristics of engineered bone.

FIG. 1.

Bioreactor design. (A) Schematic presentation of the bioreactor system used to provide perfusion through six cell-seeded scaffolds that were press-fit into the culture wells. Scaffolds were 4-mm-diameter × 4-mm-high plugs of fully decellularized, mineralized bone. (B) Computational Fluid Dynamics simulation of flow through channels indicating uniform flow to all six wells. (C) Complete experimental set-up showing bioreactor, perfusion loop, and peristaltic pump. (D) Bioreactor on a microscope stage during imaging of tissue constructs in situ. Color images available online at www.liebertonline.com/ten.

FIG. 2.

Estimates of shear stress in decellularized bone scaffolds. (A) The complex bone scaffold geometry was simplified by reducing the interconnected pore network to a bundle of parallel, cylindrical channels of varying diameters in order to estimate the shear rates. The number of channels were adjusted so that the linear velocities were equal in all cases. (B) Wall shear as a function of pore diameter and medium flow rate. Lines indicate shear values for constant linear flow rates through a scaffold 4 mm in diameter (blue) and corrected for 70% scaffold void volume (red) while varying the number of tubes. Gray area indicates range of estimated pore sizes from micro computed tomography analysis of scaffolds. Color images available online at www.liebertonline.com/ten.

FIG. 3.

Decellularized bone scaffold. (A) DNA content of native bone at various stages of preparation and after seeding with cells. (B)–(E) Hematoxylin and eosin staining of the native bone and bone scaffold. (B) Native trabecular bone. (C) Trabecular bone after washing with a high-pressure stream of water. Osteocytes are visible in the lacunae of the mineralized regions. (D) Trabecular bone after entire decellularization process showing that osteocytes are removed. (E) Central region of re-seeded bone scaffolds showing cell distribution in the pore spaces. LS, low seeding density; HS, high seeding density.

FIG. 4.

Tissue engineered bone: morphology. (A) Hematoxylin and eosin staining of seeded scaffolds after 5 weeks of culture. Cells grow along the periphery of scaffolds in all groups. Uniformity is greatest in the low seeding density, high flow (LS-HF) group, where cells grow 800 μm or more into the scaffold from the upper and lower edges, and least in the high seeding density, low flow (HS-LF) group, with growth up to approximately 350 μm. (Scale bar = 1 mm) (B) Cell density in specific regions differs significantly between groups. The LS-HF and HS-LF groups have high localized cell densities, whereas the LS-LF group has low cell density (scale bar = 200 μm) (C) Low-magnification scanning electron microscopy (SEM) images (30 ×) of external surfaces of tissue constructs showing cell growth and extracellular matrix deposits in seeded scaffolds (scale bar = 1 mm). (D) High-magnification SEM images (1000 ×) of inner regions showing cell interaction with the mineralized walls and formation of three-dimensional networks in the seeded groups (scale bar = 50 μm). (E) Micro computed tomography images of tissue constructs from all groups after 5 weeks of culture (scale bar = 1 mm). Color images available online at www.liebertonline.com/ten.

FIG. 5.

Tissue-engineered bone: marker expression. (A) Alkaline phosphatase activity of cells within the tissue constructs. (B) Total protein content. (C) DNA content. (D) Bone volume fractions. (n = 4 per group; ^*statistically different from unseeded group; ^†statistically different from all other groups). (E) Expression of bone sialoprotein and collagen I after 5 weeks of culture (scale bar = 1 mm).