PMC

Characterization of the biological apatite secreted by bmMSC-5 and bmMSC-5-IGF-1 cells in the bioreactor. After 21 days of incubation, the scaffolds were removed, and the media were centrifuged to collect the precipitates. The precipitates were dried and subjected to XRD examination using hydroxyapatite as a standard (A). The precipitates were also analyzed using FT-IR for the presence of phosphate, carbonate, and hydroxyl groups (B).

Live/Dead staining of bmMSC cell clusters in calcium-alginate scaffolds. The scaffolds seeded with bmMSC-5 or bmMSC-5-IGF-1 cells were retrieved from bioreactor at the time indicated, and stained with calcein AM (A1, B1, C1, D1, E1, F1, G1, and H1) or with PI (A2, B2, C2, D2, E2, F2, G2, and H2). The merged images (A3, B3, C3, D3, E3, F3, G3, and H3) and the calculated percentage of live and dead cells (A4, B4, C4, D4, E4, F4, G4, and H4) were shown.

The morphology of bmMSC-5 cells under static and dynamic culture conditions. (A) The morphology of bmMSC-5 cells in 2-dimentional culture plates in static culture condition was examined by light microscopy. Representative photo is shown. (B) The morphology of bmMSC-5 cells in calcium-alginate scaffolds incubated in 2-dimentional culture plates in static culture condition for 7, 14, and 21 days were examined by SEM. Representative photos are shown. (C, D) bmMSC-5 and bmMSC-5-IGF-1 cells were seeded into calcium-alginate scaffolds and kept in static state to stabilize cells in 3-dimentional environment for 24 h, and then transferred to dynamic perfused bioreactor. The morphology of bmMSC-5 and bmMSC-5-IGF-1 cell clusters in scaffolds incubated in bioreactor for 1, 7, 14, and 21 days was examined by SEM. Representative photos are shown.

Effect of IGF-1 overexpression to the osteoblastic differentiation of 3-dimentional bmMSC-5 cultures. Scaffolds which contained either bmMSC-5 or bmMSC-5-IGF-1 cells, and were incubated under osteoblastic induction in bioreactor were collected at days 7 and 14 for total RNA preparation. The levels of Runx2, alkaline phosphatase (ALP) and β-actin mRNAs were measured by RT-qPCR analyses. Normalized signals were compared to those of bmMSC-5 cells of day 7 (to which a value of one was assigned). Data represent the mean ± S.D. from three experiments. ^*, p < 0.05; ^**, p < 0.005; ^***, p < 0.001; ^#, p = 0.585 versus corresponding bmMSC-5 control.

Effect of IGF-1 overexpression to the proliferation and osteoblastic differentiation of aging bmMSCs. (A) RT-qPCR analyses. BmMSC-5 and bmMSC-8 cells were infected with Lenti virus for IGF-1 overexpression. The expression of IGF-1 mRNA in bmMSC-5, bmMSC-5-IGF-1, bmMSC-8, and bmMSC-8-IGF-1 cells was measured. (B) Cell proliferation assays. BmMSC-5, bmMSC-5-IGF-1, bmMSC-8, bmMSC-8-IGF-1 cells were cultured in media containing 2.5% of serum. Cells were harvested and counted at the time indicated. Relative cell proliferation was calculated by comparing the cell number at days 4, 8, and 12 to that of day 0 (to which a value of one was assigned). Data represent the mean ± S.D. from three experiments. ^*, p < 0.01 versus corresponding control.

Size distribution, biomineralization and cell numbers of bmMSC cell clusters. (A) The sizes of cell clusters in the scaffolds incubated in bioreactor for 1, 7, 14, and 21 days were measured by SEM (n = 20-35 for each condition, the details were revealed in supplementary Fig. ). (B, C) The cell clusters were stained with xylenol orange to indicate the biomineralized areas, and with Hoechst 33342 to indicate the location of nucleus. (D) The relative cell volume of bone-like tissues after 7, 14, and 21 days' cultivation were analyzed via F-actin Staining and calculated by Imaris software (normalized with Day 1); and (E) represented cell numbers in different size of bone-like tissues at every time points (the details were revealed in supplementary Table ). The values of cell cluster size distribution were mean ± S.D.; *p < 0.05, **p < 0.01.

Aging-related changes in the mitogenic response of human bmMSCs to IGF-1. The bmMSCs derived from 4 adult and 10 aged human donors were subjected to BrdU incorporation analyses with or without concomitant treatment of varying doses of IGF-1 as indicated. (A) The average OD450 values of adult and aged group cultured without IGF-1 treatment are shown. The difference between the groups was analyzed by the Student's t-test. (B) Relative DNA synthesis in adult and aged cells was calculated by comparing the OD450 readings from the IGF-1-treated cells to those of the untreated adult and aged control (to which a value of 1 was assigned), respectively. Data represent the mean ± S.D. from three experiments. A one-way ANOVA plus Scheffe's post hoc tests were used to analyze the differences among the untreated and IGF-1-treated groups. ^*, p < 0.05 versus untreated adult control. ^#, p < 0.05 versus untreated aged control. Comparison of the fold-change of DNA synthesis between the adult and aged groups was conducted by Student's t-test.

The effects of IGF-1 to the osteoblastic differentiation of human bmMSCs. (A) Osteoblastic differentiation. BmMSC-5 cells were induced to undergo osteoblastic differentiation with or without concomitant treatment of varying doses of IGF-1 as indicated. Cells were stained with Alizarin Red S at the days as indicated. The stains were quantitated. All of the OD₅₉₅ readings of cells were compared to that of the untreated cells of day 19 (to which a value of 1 was assigned). Data represent the mean ± S.D. from three experiments. A one-way ANOVA plus Scheffe's post hoc tests were used to analyze the differences. ^*, p < 0.05 versus the untreated cells of day 19. (B) RT-qPCR analyses. The bmMSCs from 10 aged donors were induced to the osteoblastic lineage without or with concomitant treatment of 50 and 250 ng/ml IGF-1 for 21 days. Cellular levels of alkaline phosphatase (ALP), collagen I (Col), osteopontin (OP), and β-actin mRNAs were measured. Normalized signals from cells treated with IGF-1 were compared to those of cells without IGF-1 treatment (to which a value of 1 was assigned). Data represent the mean ± S.D. from three experiments. ^*, p < 0.05 versus corresponding control. (C) Osteoblastic differentiation. bmMSC-1, -2, and -3 from adult donors and bmMSC-5, -6, and-7 from aged donors were induced to undergo osteoblastic differentiation with or without concomitant treatment of varying doses of IGF-1 as indicated. Cells were stained with Alizarin Red S at the days as indicated. All the OD₅₉₅ readings of cells were compared to that of the untreated adult cells at each time point (to which a value of 1 was assigned). Data represent the mean ± S.D. from three experiments. A one-way ANOVA plus Scheffe's post hoc tests were used to analyze the differences. ^*, p < 0.05 versus untreated adult control. Comparison of the fold-change between the adult and aged groups was conducted by Student's t-test.