Evaluation of Various Seeding Techniques for Culturing Osteogenic Cells on Titanium Fiber Mesh

Abstract

The objective of the present study was to learn more about the effect of seeding and loading techniques on the osteogenic differentiation in vitro of rat bone marrow cells into titanium fiber mesh. This material was used as received or subjected to glow discharge treatment (RFGD). The seeding methods that were used included a so-called droplet, cell suspension (high and low cell density), and rotating plate method. Osteogenic cells were cultured for 4, 8, and 16 days into titanium fiber mesh. DNA, osteocalcin, scanning electron microscopy (SEM) analysis, and calcium measurements were used to determine cellular proliferation and differentiation. DNA analysis of the differently seeded specimens showed that proliferation proceeded faster in the first versus second run for droplet and cell suspension samples. No clear and distinct additional effect was found when RFGD treatment was used. Statistical analyses revealed that high cell density and low rotational speed resulted always in a significantly higher DNA content. Calcium measurements and osteocalcin analysis showed that using high cell densities during inoculation of the scaffolds prevented the occurrence of differences between experimental runs. SEM examination showed that for droplet and cell suspension samples cells were present at only one side of the mesh. The mesh side where the cell sheet was observed depended on the additional use of glow discharge treatment. On these materials, the cells had penetrated through the meshes and formed a cell sheet at the bottom side. When rotation was used, no cell sheet was formed and cells had invaded the meshes and were growing around the titanium fibers. On the basis of our results, we conclude that (1). titanium fiber mesh is indeed suitable to support the osteogenic expression of bone marrow cells, and (2). changing the initial cell density as well as the use of dynamic seeding methods can influence the osteogenic capacity of the scaffold.

Growing Bone Tissue-Engineered Niches with Graded Osteogenicity: AnIn VitroMethod for Biomimetic Construct Assembly

“…30 However, after achieving in vitro terminal osteodifferentiation, including a large production of the calcium matrix in the short timeframe of a few weeks, osteoblast viability and synthetic activity are usually exhausted. On the other hand, naturally occurring osteogenesis results from the coexistence of MSC-derived progenies at distinct differentiative stages, which form a complex and intercommunicating microenvironment with their ECM.…”

“…The importance of finding appropriate methods for MSC seeding has also been pointed out by several authors looking for optimization of their tissue constructs; 14,30,32 however, the seeded cell number should always be considered a decisive parameter to be taken into account, as it affects the cell loss at seeding (i.e., the real cell density achieved per scaffold area) together with the scaffold-specific architecture and material and surface properties. 33 Cell-cell signals have indeed proved to be extremely important in cell proliferation, migration, and ECM molecule deposition and either too high or too low densities may be equally detrimental to achieve the desired neotissue formation.…”

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

“…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.…”

Dedifferentiated fat cells differentiate into osteoblasts in titanium fiber mesh

“…Van den Dolder and colleagues reported on the utility of the combination of TFM and BMSCs for bone tissue engineering (Van Dolder et al 2002, 2003a. In their previous studies, various techniques were used to show that cell seeding into TFM could be achieved by rotating a 10 mL tube containing a cell suspension on a rotation plate at 2 rpm (Van Dolder et al 2002, 2003b), a droplet technique in which the cell suspension was placed as a droplet on top of the TFM (Van Den Dolder et al 2003c), a cell suspension technique with the TFM in a 24 well plate (containing cell suspension) that was manually shaken every half hour (Van Den Dolder et al 2003c) and a flow perfusion culture system (Van Den Dolder et al 2003a). However, the rotation plate, droplet and cell suspension techniques showed limited cell penetration into TFM (Van Den Dolder et al 2003a, c).…”

“…It was proposed that this variance was due to BMSCs being a heterogeneous cell population, and a homogeneous cell population is necessary to produce reliable and standardized tissueengineered bone constructs (Van Dolder et al 2002, 2003c. DFAT cells comprise a highly homogeneous cell population because DFAT cells originate from a fraction of highly homogeneous mature adipocytes.…”