In vitro characterization of three-dimensional scaffolds seeded with human bone marrow stromal cells for tissue engineered growth of bone: mission impossible? A methodological approach.
ABSTRACT The aim of the present report was to evaluate current methods of in vitro analysis of three-dimensional (3D) scaffolds seeded with human bone marrow stromal cells (hBMSCs) from six bone marrow aspirates for tissue engineered growth of bone.
A series of experiments was conducted to compare methods of cell expansion and to validate analysis of proliferation and differentiation of hBMSCs in long term cultures of up to 40 days in 3D scaffolds of calcium carbonate (CaCO(3)) and mineralized collagen. Proliferation within the seeded scaffolds was monitored using cell counting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), neutral red (NR) and DNA fluorescence assays and compared with empty controls. Differentiation was assessed by means of ELISA for osteocalcin (OC) and real time PCR for OC and collagen I (Coll I).
The results showed that the scaffold differed in seeding efficacy (CaCO(3): 53.3%, min. Coll.: 83.3%). The precise identification of the number of cells in biomaterials by MTT, NR and DNA assays was problematic, as MTT and NR assay overestimated the number of cells, whereas DNA assay grossly underestimated the number of cells on the scaffolds. Monitoring of changes over time may be biased by unspecific material-dependent background activity that has to be taken into account. Identification of osteogenic differentiation is not reliable by identifying osteogenic markers such as OC in the supernatant but has to be done on the transcriptional level.
It is concluded that monitoring of in vitro procedures for the construction of biohybrid scaffolds requires more emphasis in order to make the cell based approach a reliable treatment option in tissue engineering.
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ABSTRACT: As three-dimensional (3D) cell culture systems gain popularity in biomedical research, reliable assays for cell proliferation within 3D matrices become more important. Although many cell quantification techniques have been established for cells cultured on nondegradable plastic culture dishes and cells suspended in media, it is becoming increasingly clear that cell quantification after prolonged culture in 3D polymeric scaffolds imposes unique challenges because the added presence of polymeric materials may contribute to background signal via various mechanisms including autofluorescence, diffusion gradients, and sequestering effects. Thus, additional steps are required to ensure complete isolation of cells from the 3D scaffold. The diphenylamine assay isolates cellular DNA, degrades the polymeric matrix materials, and reacts with the DNA to yield a colorimetric response. Thus, we report here a practical modification of the diphenylamine assay and show that the assay quantifies cells in 3D polyester scaffolds reliably and reproducibly as long as the necessary amount of the acidic working reagent is present. Our study also demonstrates that the sensitivity of the assay can be optimized by controlling the dimensions of the sampling volume. Overall, the DPA assay offers an attractive solution for challenges associated with 3D cell quantification.Journal of Biomedical Materials Research Part B Applied Biomaterials 12/2009; 92(2):499-507. · 2.31 Impact Factor
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ABSTRACT: The aim of this study was to develop a method for the determination of the three-dimensional (3D) distribution of cells in mineralized scaffolds and to compare the effect of two different methods of cell seeding of human bone marrow stroma cells (hBMSCs) in long-term cultures. hBMSCs were seeded into CaCO(3) scaffolds by droplet seeding using culture medium with and without the addition of fibrin. After 2, 7, 14, and 21 days of culture, the constructs were embedded into methylmethacrylate and serially sectioned using undecalcified thick section technology. Sections were serially scanned from the surface to the bottom of the scaffolds and DAPI-stained cells were automatically counted in each section using structured illumination fluorescence microscopy (FM) with serial optical sectioning and image analysis software. The data showed that the seeding efficiency was significantly higher in the scaffolds seeded with the addition of fibrin. Moreover, the number of cells increased to higher levels and remained higher for longer periods with the use of the fibrin matrix, whereas cells seeded in the medium suspension exhibited a sharp decrease after the first week of cultivation. There were distinct differences in the 3D cell distribution between the center and the periphery of the scaffolds. The use of a fibrin matrix was associated with a more uniform cell distribution 1 and 2 weeks after seeding in different levels (center vs. periphery: P>0.05), whereas cells in the medium solution group accumulated at the periphery of the scaffolds. In conclusion, automated serial optical sectioning using structured illumination FM can assess cell numbers and the 3D distribution of hBMSCs in mineralized scaffolds. This allows for a detailed analysis of the effect of different in vitro procedures used for cell seeding. The use of fibrin during seeding increases seeding efficiency and enhances both proliferation and cell survival in the central parts of the scaffolds.Clinical Oral Implants Research 12/2009; 21(2):182-8. · 3.43 Impact Factor
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ABSTRACT: The aim of the present study was to test the hypothesis that both scaffold material and the type of cell culturing contribute to the results of in vivo osteogenesis in tissue-engineered constructs in an interactive manner. CaCO3 scaffolds and mineralized collagen scaffolds were seeded with human trabecular bone cells at a density of 5 x 10(6) cells/cm(3) and were left to attach under standard conditions for 24 h. Subsequently, they were submitted to static and dynamic culturing for 14 days (groups III and IV, respectively). Dynamic culturing was carried out in a continuous flow perfusion bioreactor. Empty scaffolds and scaffolds that were seeded with cells and kept under standard conditions for 24 h served as controls (groups I and II, respectively). Five scaffolds of each biomaterial and from each group were implanted into the gluteal muscles of rnu rats for 6 weeks. Osteogenesis was assessed quantitatively by histomorphometry and expression of osteocalcin (OC) and vascular endothelial growth factor (VEGF) was determined by immunohistochemistry. CaCO3 scaffolds exhibited 15.8% (SD 3.1) of newly formed bone after static culture and 22.4% (SD 8.2) after dynamic culture. Empty control scaffolds did not show bone formation, and scaffolds after 24 h of standard conditions produced 8.2% of newly formed bone (SD 4.0). Differences between the controls and the scaffolds cultured for 14 days were significant, but there was no significant difference between static and dynamic culturing. Mineralized collagen scaffolds did not show bone formation in any group. There was a significant difference in the expression of OC within the scaffolds submitted to static versus dynamic culturing in the CaCO3 scaffolds. VEGF expression did not show significant differences between static and dynamic culturing in the two biomaterials tested. It is concluded that within the limitations of the study the type of biomaterial had the dominant effect on in vivo bone formation in small tissue-engineered scaffolds. The culture period additionally affected the amount of bone formed, whereas the type of culturing may have had a positive effect on the expression of osteogenic markers but not on the quantity of bone formation.Journal of Biomedical Materials Research Part A 08/2009; 90(2):429-37. · 2.83 Impact Factor