Cell biological evaluation of the Collagen Cell Carrier (CCC)

In vitro assays were performed with different cell populations to evaluate the biocompatibility of the collagen scaffold and its impact on cell viability and proliferation.

Murine osteoblasts and mesenchymal stem cells isolated from human adult bone marrow were cultivated on collagen scaffolds for up to 6 weeks. Osteogenic differentiation of stem cells could be induced within 4 weeks and demonstrated on the basis of the cells' time-dependent alkaline phosphatase and mineralization activity.

In a second approach, the biocompatibility of the matrix was tested using primary cardiomyocytes prepared from fetal murine hearts. After seeding, the cardiomyocytes attached to the collagen substrate within one day and maintained their spontaneous contractile activity in culture over the entire 7-day observation period.

The influence of the cell carrier on cell proliferation and viability was quantified in BrdU-incorporation studies and MTT assays using the human osteosarcoma cell line Saos‑2. No significant differences in proliferation or viability were observed between cells seeded on conventional uncoated culture plates and on those grown on the collagen foil.

Preliminary implantation studies were carried out to analyze the biocompatibility of the collagen-based scaffold in vivo. Four weeks after implantation, no immunoreactivity could be detected in histological sections of the host tissue.

Both the demonstrated biocompatibility and the excellent mechanical characteristics of this collagen carrier highlight its suitability for in vitro studies and in vivo applications in basic research and in the field of regenerative medicine.

Figures

Fig. 1.
Collagen-based scaffold; Scale bar: 10 mm. Fig. 2.
Detection of alkaline phosphatase activity (black staining) of differentiating osteoblasts. Cells were culured on the collagen carrier for 14 days. Scale bar: 100 µm.

Fig. 3.
Fetal cardiomyocytes cultured for 7 days on the collagen scaffold. Immunofluorescence staining for alpha-actinin (red) demonstrating the typically arranged Z-discs. Nucleus was labelled with DAPI (blue). During the culture period cardiomyocytes maintained their spontaneous contractile activity. Scale bar: 20 µm.

Fig. 4.
Proliferation assay with the human osteosarcoma cell line Saos-2. Immunofluorescence staining of BrdU-positive cells (red nuclei) cultured on conventional culture plates (A) and on the collagen carrier (B) for 7 days. BrdU-negative nuclei show blue DAPI fluorescence. Scale bar: 100 µm.

Fig. 5.
Influence of the cell carrier on cell proliferation and viability of Saos-2 cells as quantified by BrdU incorporation (A) and MTT assays (B). For analysis purposes, the cells were cultured for 7 days on conventional uncoated culture plates and on the collagen scaffold. Data are expressed as mean ± SD; n=5. There were no significant differences in cell proliferation activity and viability between the two culture conditions.

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	Fig. 3. Fetal cardiomyocytes cultured for 7 days on the collagen scaffold. Immunofluorescence staining for alpha-actinin (red) demonstrating the typically arranged Z-discs. Nucleus was labelled with DAPI (blue). During the culture period cardiomyocytes maintained their spontaneous contractile activity. Scale bar: 20 µm.
		Fig. 4. Proliferation assay with the human osteosarcoma cell line Saos-2. Immunofluorescence staining of BrdU-positive cells (red nuclei) cultured on conventional culture plates (A) and on the collagen carrier (B) for 7 days. BrdU-negative nuclei show blue DAPI fluorescence. Scale bar: 100 µm.
		Fig. 5. Influence of the cell carrier on cell proliferation and viability of Saos-2 cells as quantified by BrdU incorporation (A) and MTT assays (B). For analysis purposes, the cells were cultured for 7 days on conventional uncoated culture plates and on the collagen scaffold. Data are expressed as mean ± SD; n=5. There were no significant differences in cell proliferation activity and viability between the two culture conditions.