Controlled Cell-Seeding Methodologies: A First Step Toward Clinically Relevant Bone Tissue Engineering Strategies

Abstract

The repair of large and complex bone defects could be helped by a cell-based bone tissue engineering strategy. A reliable and consistent cell-seeding methodology is a mandatory step in bringing bone tissue engineering into the clinic. However, optimization of the cell-seeding step is only relevant when it can be reliably evaluated. The cell seeding efficiency (CSE) plays a fundamental role herein. Results showed that cell lysis and the definition used to determine the CSE played a key role in quantifying the CSE. The definition of CSE should therefore be consistent and unambiguous. The study of the influence of five drop-seeding-related parameters within the studied test conditions showed that (i) the cell density and (ii) the seeding vessel did not significantly affect the CSE, whereas (iii) the volume of seeding medium-to-free scaffold volume ratio (MFR), (iv) the seeding time, and (v) the scaffold morphology did. Prolonging the incubation time increased the CSE up to a plateau value at 4 h. Increasing the MFR or permeability by changing the morphology of the scaffolds significantly reduced the CSE. These results confirm that cell seeding optimization is needed and that an evidence-based selection of the seeding conditions is favored.

Three-Dimensional Characterization of Tissue-Engineered Constructs by Contrast-Enhanced Nanofocus Computed Tomography

“…Before cell seeding, all scaffolds were prewetted by vacuum impregnation in a cell culture medium for 2 h in a humidified incubator at 37°C, and dried overnight in a nonhumidified incubator. 38 Standard 2D hPDC culture hPDCs were isolated from periosteal biopsies of different donors as described previously. 39 This procedure was approved by the ethics committee for Human Medical Research (KU Leuven) and with patient informed consent.…”

“…38 In vitro cell culture in the TE constructs lasted for 14, 21, and 28 days under static (n = 7) or dynamic (n = 7) culture conditions. For static culture, TE constructs were positioned in 12-well plates (Greiner Bio One) containing a 3 mL cell culture medium, and incubated at 37°C in a humidified and CO 2 -controlled incubator (relative humidity: 95%, 5% CO 2 ).…”

“…Constructs were rinsed with 1 mL PBS, incubated in the staining solution (0.5 mL calcein AM and 2 mL ethidium homodimer in 1 mL PBS) for 20 min under normal cell culture conditions, and finally imaged using a Leica M165 FC microscope. 38 …”

Noninvasive Real-Time Monitoring by AlamarBlue®DuringIn VitroCulture of Three-Dimensional Tissue-Engineered Bone Constructs

“…Before cell seeding, all scaffolds were prewetted with a cell culture medium consisting of Dulbecco's modified Eagle's medium with high-glucose (Invitrogen) containing 10% fetal bovine serum (BioWhittaker), 1% sodium pyruvate (Invitrogen), and 1% antibiotic-antimycotic (100 units/mL penicillin, 100 mg/mL streptomycin, and 0.25 mg/mL amphotericin B; Invitrogen) by vacuum impregnation in the cell culture medium for 2 h in a humidified incubator at 37°C, and drying overnight in a nonhumidified incubator. 31 …”

“…The TE constructs were additionally characterized in a destructive way by a combined and validated end-point analysis as an established reference for the AB measurements: a qualitative live/dead staining to assess the viability and homogeneity, and a quantitative DNA analysis technique to assess the total cell number. 30,31 Resazurin based real-time analyses were already performed on 3D cell cultures but so far not correlated to established quantitative techniques for the determination of cell number. 28 This study evaluated three different aspects of the AB metabolic assay to assess its potential as noninvasive, realtime monitoring tool for bioreactor culture of TE constructs: (1) the influence of the AB incubation time on the measured metabolic activity, (2) the correlation between the metabolic activity and the cellularity upon cell seeding, and (3) the correlation between the metabolic activity and the cellularity upon TE construct culturing.…”