Collagen type I hydrogel allows migration, proliferation, and osteogenic differentiation of rat bone marrow stromal cells

Abstract

Hydrogels are potentially useful for many purposes in regenerative medicine including drug and growth factor delivery, as single scaffold for bone repair or as a filler of pores of another biomaterial in which host mesenchymal progenitor cells can migrate in and differentiate into matrix-producing osteoblasts. Collagen type I is of special interest as it is a very important and abundant natural matrix component. The purpose of this study was to investigate whether rat bone marrow stromal cells (rBMSCs) are able to adhere to, to survive, to proliferate and to migrate in collagen type I hydrogels and whether they can adopt an osteoblastic fate. rBMSCs were obtained from rat femora and plated on collagen type I hydrogels. Before harvest by day 7, 14, and 21, hydrogels were fluorescently labeled, cryo-cut and analyzed by fluorescent-based and laser scanning confocal microscopy to determine cell proliferation, migration, and viability. Osteogenic differentiation was determined by alkaline phosphatase activity. Collagen type I hydrogels allowed the attachment of rBMSCs to the hydrogel, their proliferation, and migration towards the inner part of the gel. rBMSCs started to differentiate into osteoblasts as determined by an increase in alkaline phosphatase activity after two weeks in culture. This study therefore suggests that collagen type I hydrogels could be useful for musculoskeletal regenerative therapies.

Figure 1

Qualitative analysis of migration, and proliferation of rBMSCs in collagen type I hydrogels during a three weeks period. Collagen type I hydrogels were seeded with rBMSCs. Hydrogels (n=3) were labeled with Calcein AM/Ethidium homodimer-1 prior to analysis by day 7 (A), day 14 (B), and day 21 (C). For fluorescent microscopy, cell-matrix constructs were immediately frozen and cryo-cut in saggital sections. Cells are shown in red. The image demonstrates the increase of the overall cell number and the progressive increase in the migration distance of some rBMSCs in collagen type I hydrogels. The cell migration front is highlighted with a white dotted line. Representative images were taken at 100x magnification.

Figure 2

Quantitative analysis of the distance of rBMSC migration in collagen type I hydrogels during three weeks of culture. Collagen type I hydrogels were overlaid with rBMSCs and cultured for 21 days. Prior to analysis, samples were labeled with a fluorescent dye and cryo-cut. The distance between each cell and the hydrogel surface was determined by standardized histomorphometric analysis. Shown are the migration distances after 7 days, 14 days, and 21 days. Data represent means ± standard deviation for three samples (n=3, 6 sections/sample). *Indicates statistical significance compared to day 7 (p<0.05), and # indicates statistical significance compared to day 14 (p<0.05).

Figure 3

Viability of rBMSCs in collagen type I hydrogels. Collagen type I hydrogels (n=3) were seeded with rBMSCs and incubated for 21 days. Prior optical analysis by laser scanning confocal microscopy, native cell-matrix constructs were labeled with Calcein AM/Ethidium homodimer-1. Shown are representative images taken by day 21 in two different magnifications (A=100x, B=400x). Viable cells appear in green.

Figure 4

Proliferation of rBMSCs and relation of migrated to non-migrated rBMSCs after plating on collagen type I hydrogels. Cell-seeded hydrogels were cultured for a period of 21 days. Samples were fluorescently labeled and cryo-sectioned by day 7, day 14, and day 21. The total number of all rBMSCs and the number of migrated cells as well as the number of cells that remained on the gel surface was individually determined by standardized histomorphometric analysis. The graph shows a time-related increase of the total and the subgroup-related cell number. Data represent means ± standard deviation for three samples (n=3, 6 sections/sample). *Indicates statistical significance compared to day 7 (p<0.05), and # indicates statistical significance compared to day 14 (p<0.05) (A). A proliferation factor was calculated based on the increase in cell number between day 7 and day 14. Shown are the proliferation factors for cells grown on hydrogels and for cells grown in control culture (B). The percentage of the migrated cells is about one third of all rBMSCs and remains unchanged during the experiment (p>0.05) (C).

Figure 4

Proliferation of rBMSCs and relation of migrated to non-migrated rBMSCs after plating on collagen type I hydrogels. Cell-seeded hydrogels were cultured for a period of 21 days. Samples were fluorescently labeled and cryo-sectioned by day 7, day 14, and day 21. The total number of all rBMSCs and the number of migrated cells as well as the number of cells that remained on the gel surface was individually determined by standardized histomorphometric analysis. The graph shows a time-related increase of the total and the subgroup-related cell number. Data represent means ± standard deviation for three samples (n=3, 6 sections/sample). *Indicates statistical significance compared to day 7 (p<0.05), and # indicates statistical significance compared to day 14 (p<0.05) (A). A proliferation factor was calculated based on the increase in cell number between day 7 and day 14. Shown are the proliferation factors for cells grown on hydrogels and for cells grown in control culture (B). The percentage of the migrated cells is about one third of all rBMSCs and remains unchanged during the experiment (p>0.05) (C).

Figure 4

Proliferation of rBMSCs and relation of migrated to non-migrated rBMSCs after plating on collagen type I hydrogels. Cell-seeded hydrogels were cultured for a period of 21 days. Samples were fluorescently labeled and cryo-sectioned by day 7, day 14, and day 21. The total number of all rBMSCs and the number of migrated cells as well as the number of cells that remained on the gel surface was individually determined by standardized histomorphometric analysis. The graph shows a time-related increase of the total and the subgroup-related cell number. Data represent means ± standard deviation for three samples (n=3, 6 sections/sample). *Indicates statistical significance compared to day 7 (p<0.05), and # indicates statistical significance compared to day 14 (p<0.05) (A). A proliferation factor was calculated based on the increase in cell number between day 7 and day 14. Shown are the proliferation factors for cells grown on hydrogels and for cells grown in control culture (B). The percentage of the migrated cells is about one third of all rBMSCs and remains unchanged during the experiment (p>0.05) (C).

Figure 5

Osteoblast differentiation of rBMSCs in collagen type I hydrogels during three weeks of osteogenic culture. Collagen type I hydrogels were seeded with rBMSCs and incubated for a period of 21 days in medium containing osteogenic supplements. Hydrogels were homogenized after 7 days, 14 days, and 21 days. Alkaline phosphatase activity was quantified and normalized to DNA. Osteogenic differentiation of rBMSCs started after two weeks of culture and led to a significantly increased level of ALP activity by day 21. Data represent means ± standard deviation for n=3. # Indicates statistical significance compared to day 14 (p<0.05).