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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences logoLink to The Journals of Gerontology Series A: Biological Sciences and Medical Sciences
. 2010 Jul 19;65A(10):1051–1059. doi: 10.1093/gerona/glq114

Fibroblast Growth Factor-2 Stimulates the Proliferation of Mesenchyme-Derived Progenitor Cells From Aging Mouse and Human Bone

Guomin Ou 1, Lyndon Charles 1, Seth Matton 2, Craig Rodner 3, Marja Hurley 4, Liisa Kuhn 1, Gloria Gronowicz 2,✉
PMCID: PMC2949335  PMID: 20643704

Abstract

The potential of fibroblast growth factor-2 (FGF-2) to stimulate osteoprogenitors in aging bone was investigated. Previous work showed a decrease in bone formation in cell cultures derived from bone of elderly female patients, but not in cells from age-matched male or younger female patients, with transforming growth factor β increasing bone formation but not increasing osteoprogenitors. In the present study, FGF-2 was shown to significantly stimulate, in a dose-dependent manner, proliferation of mesenchyme-derived progenitor cells from bones of young and old mouse and humans. In proliferation assays, human cells were more responsive to lower concentrations (0.0016 ng/mL) of FGF-2 than mouse cells, but proliferation was less in cells from older bone. Immunofluorescence microscopy revealed that FGF-2 increased and prevented the decline in cells expressing activated leukocyte cell adhesion molecule, a novel marker for early lineage osteoblasts, but not α-smooth muscle actin. FGF-2 may have therapeutic potential for stimulating osteoblast progenitors in aging.

Keywords: Aging, Bone, Cell proliferation, Fibroblast growth factor-2, Osteoblasts

THE ultimate goal of our research was to develop bone-augmenting therapies for aging humans. Fibroblast growth factors (FGFs) are known to stimulate mouse osteoprogenitor proliferation (1,2). The present study was initiated to determine if mesenchyme-derived progenitor cells isolated from the bones of humans had a comparable proliferative response with fibroblast growth factor-2 (FGF-2) and to compare this response with similarly derived cells from bones of mice. Secondly, the effect of age on number of osteoprogenitors and cell proliferation were compared in these two species. In determining an appropriate model for studying effects of FGF-2 on osteoblasts, it was important to determine if mouse and human mesenchyme-derived progenitor cells from adult bone responded in a similar manner to FGF-2 so that mice could be used as a model animal for studying FGF-2 as a therapeutic intervention in aging bone.

The FGFs are a family of heparin-binding polypeptide growth factors that are known to regulate cell proliferation and differentiation. FGFs are encoded by 23 single-copy genes and have four receptors that are expressed during human embryonic and fetal development (3). FGF receptors are important regulators of bone development and are differentially expressed in bone, cartilage, and bone marrow stromal cells (4–6). They play a significant role in a number of biologic processes, such as osteogenesis, chondrogenesis, angiogenesis, wound healing, and apoptosis (7–9). FGF-2, also known as basic FGF, is one member of this family and regulates the proliferation and differentiation of a variety of cell types including osteoblasts, increases the osteogenic phenotype of human bone marrow–derived cells, and is stored in the extracellular matrix (2,8,10–14). In contrast to inhibitory results with continuous FGF-2 treatment in vitro, FGF-2 treatment in animals and intermittent FGF-2 treatment in vitro stimulate bone formation (9,15,16). Overexpression of FGF-2 or continuous FGF-2 treatment sustained the proliferative and osteogenic potential of adipose-derived stromal cells by increasing osteoprogenitor cells but negatively regulated osteoblast differentiation (17). Disruption of the FGF-2 gene in mice led to significant loss of bone mass and bone formation, as well as downregulation of bone morphogenetic protein-2 (18–20). Although the growth plates of the mouse long bones, in which the FGF-2 gene was disrupted, were reported to show no abnormalities, additional studies showed that, as these mice aged, they developed decreased bone mass and bone formation rates suggesting a role for FGF-2 in maintaining bone mass with age (6,19). Kato and coworkers (21) observed that FGF-2 and platelet-derived growth factor were the only growth factors that increased mineralized nodule formation in calvarial osteoblasts from aged rats and FGF-2 was twice as potent as platelet-derived growth factor. Additional studies demonstrated the significant role of FGF-2 in stimulating proliferation and osteogenic expression of stromal bone marrow progenitors derived from adult rats (22). Therefore, FGF-2 may have a therapeutic value in offsetting the age-induced decline in cell and tissue activity and responsiveness. Our study was undertaken to compare the effect of FGF-2 on cells from the bones of young and old mice with the cells from the bones of young and old patients. These isolated cells from human bone had previously been shown to have the potential to become osteoblasts, adipocytes, or chondrocytes and, therefore, were coined “multipotential mesenchymal stem cells from adult bone” (23,24). The cells that are grown in culture from outgrowths of human bone also have been shown to be identical in phenotype when differentiated in culture to mesenchymal stem cells obtained from bone marrow aspirates (25,26). We consider these cells to be multipotential mesenchyme-derived progenitor cells from adult bone.

Although the biologic role of FGF-2 appears to be well defined, there are limited studies comparing mesenchyme-derived progenitor cell proliferation in young and old mice and humans. Surprisingly, direct comparison of the responsiveness of cells from these two species is difficult to find in the scientific and aging literature, although the mouse is used as a model for many human diseases. Primary mesenchyme-derived progenitor cells derived from juvenile (2-day-old) and adult (60-day-old) rat calvaria demonstrated decreased cell proliferation and differentiation in response to FGF-2 in cultures from older animals compared with younger cultures and a differential expression of FGF-2 isoforms (27). Another study compared FGF-2 gene expression in young and old mice and revealed a decline in both the number of wound-responsive FGF-2 and FGF receptor genes in the skin of aged mice (28). This change in FGF-2 gene expression could account, at least in part, for the age-related slowing of wound healing in mice. Previous work in our laboratory showed that multipotential mesenchyme-derived progenitor cells from adult bone of elderly female patients had a reduced capacity to make bone relative to age-matched cells from male patients and young female patients, although their ability to proliferate did not differ (29). The cells from elderly female patients had a decreased ability to adhere to tissue culture plastic, decreased levels of messenger RNA levels of alpha1(I) procollagen, alkaline phosphastase, and bone sialoprotein, and decreased mineralization compared with cells isolated in a similar manner from younger female patients and all ages of male patients. Transforming growth factor β was able to stimulate bone formation in cultures from elderly female patients by increasing osteoblast differentiation but did not affect the number of osteoprogenitors (30). In the present study, cells grown from female mouse and human bone were compared to determine if FGF-2 could increase the proliferation of multipotential mesenchyme-derived progenitor cells and to identify which cells were stimulated so that future studies involving mouse models might generate data relevant to human aging.

METHODS

Isolation and Culture of Mesenchyme-Derived Progenitor Cells From Adult Bone

All experiments were repeated at least three times from different isolations of mesenchyme-derived progenitor cells from young and old mouse bones and patients. The results presented as figures were similar for all experiments.

Mouse.—

Mesenchyme-derived progenitor cells were obtained from the calvaria and femurs of 3-month-old and 19- to 22-month-old female BALB/c mice. Soft tissues were removed, and the bones were aseptically harvested. The epiphyses were cut off, and the bone marrow flushed out with phosphate-buffered saline (PBS) using a syringe and needle. The femoral diaphyses were chopped into 1-mm2 fragments. Bone fragments were extensively and repeatedly washed with cold PBS to remove adherent marrow cells. Calvarial cells were obtained from frontal and parietal bones. After the calvariae were aseptically harvested, the soft tissues were scraped off, the sutures were removed, and the remaining bone was chopped into 1-mm2 fragments. Fragments were transferred to 100-mm dishes and cultured in Dulbecco's minimum essential medium/F-12 supplemented with 10% fetal bovine serum (FBS; Gibco/Invitrogen, Rockville, MD), 100 U/mL penicillin-G (Sigma-Aldrich, St. Louis, MO), and 100 μg/mL streptomycin (Sigma). Previous studies had used enzymatically digested calvaria, but we found that very few cells could be obtained with this method in the older mice; therefore, we developed this new method for comparing osteoblasts from different ages of mice.

Human.—

Bone discarded from orthopedic procedures (Institutional Review Board exempt) was obtained from young (21-, 29-, and 35-year-old) and older (56-, 68-, 72-, 74-, and two 58-year-old) female patients. After the human bones were aseptically harvested, the soft tissues were scraped off, and the remaining bone chips were chopped into 1-mm2 fragments. Human bone fragments were transferred to 100-mm dishes. Both human and mouse bone fragments were cultured in DMEM/F-12 supplemented with 10% FBS and antibiotics. Culture medium was replaced two times per week. Mesenchyme-derived progenitor cells started to migrate from the bone chips after 1–2 weeks. At near confluency on Days 10–12, cells were removed using 0.25% trypsin and 0.1% EDTA (Gibco) in PBS and plated for the proliferation and immunofluorescence experiments.

MTS Assay for Proliferation

Mesenchyme-derived progenitor cells were trypsinized and seeded at a density of 10,000/cm2 in 96-well plates in DMEM/F-12 with 10% FBS and 1% antibiotics for 4 hours. The medium was changed to vehicle or various concentrations of FGF-2 (0.0016, 0.016, 0.16, and 1.6 ng/mL) (recombinant human FGF basic, catalog number 223-FB-CF; R&D Systems, Minneapolis, MN; mitogenic activity was assessed in mouse NR6R-3T3 fibroblasts by R&D) in 0.5% heat-inactivated FBS in DMEM/F-12 for 4, 24, 48, and 72 hours. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) (CellTiter 96; Promega, Madison, WI) was used to quantify proliferation.

Immunofluorescence

Mesenchyme-derived progenitor cells were grown in the eight chambers per slide (Lab-Tek, Nunc, Naperville, IL) at the density of 10,000 cells/cm2 for mouse and 20,000 cells/cm2 for human cells. The density for the human cells was increased because they did not adhere as well as the mouse cells to the chamber slides. All cells were cultured in DMEM/F-12 with 10% FBS and 1% antibiotics for 4 hours prior to the replacement of medium with heat-inactivated 0.5% FBS and varying concentrations of FGF-2. At 4, 24, 48 and 72 hours, the cells were fixed with 2% paraformaldehyde for 60 minutes on ice, washed with PBS, and then left in 5% sucrose in PBS at 4°C until all time points were ready for processing together. The primary antibodies were rabbit polyclonal activated leukocyte cell adhesion molecule (ALCAM, sc-25624; Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-actin, α-smooth muscle-Cy3 antibody (α-smooth muscle actin [α-SMA]; Sigma C6198, Sigma), and affinity-purified goat polyclonal antibody to alkaline phosphatase (sc-23430; Santa Cruz Biotechnology). The slides were rinsed in PBS after each step. For staining α-SMA and alkaline phosphatase but not ALCAM (because it is localized to the plasma membrane), the slides were permeabilized with 0.1% Triton for 10 minutes at room temperature. Slides were rinsed in PBS and then incubated at room temperature in a blocking solution of 4% BSA in PBS for 1 hour. The primary antibodies were diluted with 4% BSA in PBS as follows: 1:100 for ALCAM and alkaline phosphatase and 1:800 for α-SMA, and the slides were incubated in a humidified chamber at 4°C overnight as well as control slides with blocking solution alone. Slides were rinsed and then incubated at room temperature for 1 hour in the dark with Fluor 555–conjugated donkey anti-goat IgG for alkaline phosphatase and rhodamine-conjugated donkey anti-rabbit IgG for ALCAM (Santa Cruz Biotechnology). The slides were extensively washed with PBS, incubated with 1 μg/mL 4′,6-diamidino-2-phenylindole dihydrochloride (Sigma) in PBS for 10 minutes to visualize cell nuclei, mounted in 2.5% n-propyl-gallate in 1:1 PBS:glycerol to prevent quenching, viewed, and photographed with a Nikon fluorescence microscope. Photomicrographs were evaluated and analyzed with ImageJ software to count the number of positive/total cells.

Statistical Analysis

Experimental data and error bars represent a mean ± standard deviation for each group. The statistical analysis of the results was performed by two-way analysis of variance with multiple comparison procedures to isolate group mean differences followed by the Bonferroni posttest with the conventional 0.05 level considered to reflect statistical significance. All experiments were repeated at least three times from different isolations of mesenchyme-derived progenitor cells from young and old mouse bones and patients with at least four samples per experimental group.

RESULTS

Varying doses of FGF-2 (0.0016, 0.016, 0.16, and 1.6 ng/mL) were tested for their ability to stimulate the proliferation of female mouse mesenchyme-derived progenitor cells from calvaria and femur and female human mesenchyme-derived progenitor cells from cancellous bone (Figure 1). Although the MTS assay is an assay for cellular metabolic activity, it is considered to reflect cell proliferation. The results of our MTS assays were compared with results with [3H]-thymidine administration for the last 4 hours of a 24-, 48-, and 72-hour culture period of human mesenchyme-derived progenitor cells. The results were similar to those with the MTS assay, demonstrating that the MTS assay is appropriate for testing the rate of cell proliferation (unpublished data). In addition, the change in the values of the MTS assay was proportional to an increase in cell number determined by counting cells in the light microscope (unpublished data). Comparison of mesenchyme-derived progenitor cells from calvaria and femurs of young animals (Figure 1A and C) demonstrated that in young mice the cells from the femur were more sensitive to FGF-2 (0.016, 0.16, and 1.6 ng/mL) and demonstrated an earlier response starting at 4 and 24 hours without an effect on mesenchyme-derived progenitor cells from young mouse calvaria at those time points. At the later time points of 48 and 72 hours, both 0.16 and 1.6 ng/mL FGF-2 stimulated proliferation. Mesenchyme-derived progenitor cells from old mouse calvaria and femurs (Figure 1B and D) did not show a significant increase from controls until 48 and 72 hours of FGF-2 treatment, and the significant increase in proliferation occurred at similar FGF-2 doses.

Figure 1.

Figure 1.

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Quantitative analysis by MTS assay of proliferation in mesenchyme-derived progenitor cells from young and old female mouse calvaria (A and B), young and old female mouse femurs (C and D), and young and old female patients (E and F) exposed to vehicle or various concentrations of fibroblast growth factor-2 (FGF-2) (ng/mL). Cell growth was stimulated in both the young and the old cell cultures in a dose-dependent manner.

A comparison of mouse versus human mesenchyme-derived progenitor cells surprisingly showed that human cells responded to lower doses of FGF-2 (0.0016 ng/mL) compared with mouse cells (Figure 1). This finding was confirmed in mesenchyme-derived progenitor cells from all three young and old patients. In addition, human cells from younger patients had a greater increase in proliferation in response to FGF-2 than cells from young mouse calvaria or femurs. For example, the increase in proliferation at 48 hours in cells from young mouse femurs treated with 0.016 ng/mL of FGF-2 was 7.5%, whereas the increase in proliferation in cells from young humans was 13.9% (Figure 1C vs Figure 1E), and this difference was even greater at 72 hours (approximately 10-fold). However, cells from both old mice and humans had a similar magnitude in response to FGF-2 (Figure 1D vs Figure 1F). Interestingly, only the cells from young human patients consistently demonstrated a decrease in proliferation at the early time points with the highest concentrations of FGF-2, but this effect diminished with time in culture with all doses of FGF-2 stimulating proliferation by 72 hours compared with vehicle-treated cultures.

To determine the phenotype of the cells that were stimulated by FGF-2 to proliferate, immunocytochemistry was performed for various differentiation markers of the osteoblast lineage. Alkaline phosphatase has been shown to be an early marker of osteoblast differentiation (31,32). The intermediate dose of 0.16 ng/ml FGF-2 was chosen for the subsequent studies. Because mesenchyme-derived progenitor cells from femoral bones were more responsive to FGF-2 than calvarial cells as seen in Figure 1, cells from femoral bones were used for all the immunofluorescence analysis. Immunofluorescence staining for alkaline phosphatase demonstrated that more that 95% of the mesenchyme-derived progenitor cells from both mouse and human bone expressed this protein (Figure 2A and B, respectively). Therefore, further quantification of FGF-2 stimulation of alkaline phosphatase–stained cells was not undertaken because most of the cells in culture were stained. However, each passage of cells was routinely stained for this marker to ensure that all our cultures from mouse and human bones were consistent in cell phenotype, and they all exhibited the same high level of staining for alkaline phosphatase throughout the experiments. As a control, blocking buffer alone and the second antibody were tested and exhibited no staining for all three tested first antibodies (data not shown).

Figure 2.

Figure 2.

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Immunofluorescence microscopy of alkaline phosphatase staining. Panel (A) is representative photograph of mesenchyme-derived progenitor cells from old female mouse femurs treated with vehicle and 0.16 ng/mL fibroblast growth factor-2 (FGF-2) for 24 hours. Panel (C) is representative photograph of cells from the bone of an elderly female human participant treated with vehicle and 0.16 ng/mL FGF-2 for 24 hours. More than 95% of the cells stained for alkaline phosphatase in both the mouse and the human cell cultures.

ALCAM, a member of the IgG superfamily ligand for CD6, has been found in osteogenic precursors from bone marrow (33) and was therefore localized in the mouse and human cultures by immunocytochemistry. Approximately 20% of the mesenchyme-derived progenitor cells from young mice stained for ALCAM at 24 hours and 0.16 ng/mL of FGF-2 significantly increased positively stained cells by 36% (p > .05) (Figure 3A). No difference in the percentage of stained cells was found at 72 hours as the percentage of ALCAM-stained cells appeared to decrease with time in culture. Because the number of cells increased with time in culture by MTS assay (Figure 1), these data suggest that the ALCAM+ cells were losing the expression of this early preosteoblast marker as they proliferate and differentiate in culture. In the mesenchyme-derived progenitor cells derived from old mice, the increase in ALCAM+ cells was seen later at 72 hours and was greater, 78%, than that seen in the young cells (Figure 3B). Mesenchyme-derived progenitor cells derived from young patients (32-year-old female) were similar to the cells from the old mouse in that they demonstrated a significant increase at 72 hours of 42% and demonstrated a decline in ALCAM+ cells with time in culture (Figure 4A). However, mesenchyme-derived progenitor cells from the illustrated 68-year-old female patient (Figure 4B) had increased ALCAM staining at 24 and 72 hours. Interestingly, there were fourfold more ALCAM+ cells in untreated mesenchyme-derived progenitor cells from humans than from mouse with increased responsiveness to FGF-2 in humans compared with mice.

Figure 3.

Figure 3.

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Immunofluorescence microscopy of activated leukocyte cell adhesion molecule (ALCAM) staining of mesenchyme-derived progenitor cells from young (A) and old (B) mouse femoral bones. Cells were treated with vehicle or 0.16 ng/mL fibroblast growth factor-2 (FGF-2) for 24 and 72 hours, and representative fields of the staining are shown along with graphs of the results. Approximately 20% of the cells from young mice stained for ALCAM at 24 hours, and 0.16 ng/mL FGF-2 significantly increased the percentage of positively stained cells. In the cell cultures derived from old mice, the increase in ALCAM-expressing cells was seen at 72 hours but not at 24 hours of culture.

Figure 4.

Figure 4.

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Immunofluorescence microscopy of activated leukocyte cell adhesion molecule staining of mesenchyme-derived progenitor cells from the bone of young (A) and old (B) female human participants. Cells were treated with vehicle or 0.16 ng/mL fibroblast growth factor-2 (FGF-2) for 24 and 72 hours, and representative fields of the staining are shown along with graphs of the results. In (A) and (B), ALCAM+ cells derived from a young female patient (32 years old) and old female patient (68 years old), respectively, significantly declined with time in control cultures. However, in (A), 0.16 ng/mL FGF-2 prevented the decline in ALCAM+ cells with a 42% increase in stained cells at 72 hours compared with the control cultures at 72 hours. In (B), FGF-2 increased ALCAM+ cells at both 24 and 72 hours in mesenchyme-derived progenitor cells from this elderly patient.

A relatively new marker of the osteoblast lineage is α-SMA that is expressed in cells with a myofibroblast/pericyte phenotype, which are considered to have the capability to form osteoblasts (34). Therefore, immunocytochemistry was used to determine if FGF-2 stimulated the number of cells expressing α-SMA. No changes in the percentage of α-SMA-stained cells were found with FGF-2 treatment in either young or old mouse (Figure 5A) or young or old (Figure 5B) human mesenchyme-derived progenitor cell cultures. However, human cells had more than twofold more α-SMA+ cells (approximately 20%) than mouse cell cultures (approximately 10%), but no significant differences in α-SMA+ cells were found with age in either species. In the untreated human mesenchyme-derived progenitor cell culture, there was a significant increase in α-SMA+ cells from 24 to 72 hours (Figure 5B), but the increase was not significant in mouse mesenchyme-derived progenitor cells.

Figure 5.

Figure 5.

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Immunofluorescence microscopy of smooth muscle actin (SMA) staining of mesenchyme-derived progenitor cells from mouse femoral bones (a) and human female patients (b) from young (A) and old (B) participants. Cells were treated with vehicle or 0.16 ng/mL fibroblast growth factor-2 (FGF-2) for 24 and 72 hours. Representative fields of the staining are shown along with graphs of the results. No significant differences in α-SMA staining were found with FGF-2. However, in cells from young mice, there was a significant increase in α-SMA-stained cells in the vehicle-treated group with time.

DISCUSSION

FGF-2 was shown to stimulate, in a dose-dependent manner, the proliferation of mesenchyme-derived progenitor cell cultures derived from adult bone. Although FGF-2 has previously been shown to be anabolic for osteoblast progenitor proliferation (1,2,15), this report is the first comparison of FGF-2 responsiveness between young and old, mouse and human, mesenchyme-derived progenitor cells from bone. Similarities between species were found in the dose–response curve to FGF-2, with aging diminishing the responsiveness to FGF-2. Previous studies have used calvaria digests to obtain bone cells, but we had a very poor and variable yield of cells from older mouse bones. In this study, we developed a new method for obtaining these cells by mincing bones from calvaria and femurs and allowing cell outgrowth, which is the same method that we use for human bone samples. Thus, cells cultured from mouse bone appear to be a good model in which to study some of the aging processes found in human bone cells. In particular, FGF-2 may have a therapeutic potential for augmenting bone formation in fracture healing in elderly patients (35) that could be studied in the mouse.

Although cells from older bones were less responsive to FGF-2 than those from younger bone in both species, mesenchyme-derived progenitor cells from human bone increased their proliferation at lower doses of FGF-2 than mouse, demonstrating greater sensitivity to FGF-2. Because it was shown that it is the ALCAM-expressing population of cells that increase upon FGF-2 administration, this finding of greater sensitivity may be due to human cultures having higher levels of ALCAM expression than mouse. In fact, there were fourfold more ALCAM+ cells in the human mesenchyme-derived progenitor cell cultures than in those of mice. There have only been a few reports of CD166/ALCAM, a ligand also of the T-cell molecule CD6, expression by osteoblasts (33,36,37). Mesenchyme-derived progenitor cells from the perichondrium have also been shown to express ALCAM (38). In the present study, ALCAM was expressed at a similar level in both young and old cell cultures within each species; however, there were different time lines in the response to FGF-2. The osteoblast progenitors express both ALCAM and alkaline phosphatase because both markers were seen in greater than 80% of the cell cultures. However, ALCAM+ cells rapidly declined with time in culture, but alkaline phosphatase staining did not change during 72 hours of culture, suggesting that alkaline phosphatase expression did not identify the osteoprogenitor pool of cells that respond initially to FGF-2. FGF-2 appeared to stimulate the proliferation of the ALCAM+ cells in the cell cultures from old human bone and also maintain their number in cell culture in both human and mouse cell cultures because ALCAM+ cell numbers remained elevated at 72 hours. In assessing the percentage of untreated cells initially expressing alkaline phosphatase, ALCAM, and α-SMA, we found no significant age-related differences in the percentage of cells expressing these proteins. However, ALCAM+ cells in older mice and human retained their ability to respond to FGF-2 in a similar manner to bone cells from younger mice and patients, supporting the possible role of therapeutic potential of FGF-2 in aging bone. Additional studies dealing with long-term effects of FGF-2 on the ALCAM+ cell population in bone formation are being undertaken in vitro and in vivo. Another interesting finding was that the higher concentrations of FGF-2 inhibited proliferation at the early time points only in cell cultures from young female patients. At the present time, it is not known why this occurred except to speculate that it may be due to estrogen status of these patients because there is a recent report in endothelial and MCF-7 cells that some of actions of FGF-2 are dependent on the estrogen receptor (39).

Alpha-SMA is a relatively new marker of osteoprogenitor cells (34) and is found in myofibroblasts/pericytes that are located in bone, which is highly dependent on blood vessels for bone development. In our study, both human and mouse mesenchyme-derived progenitor cell cultures expressed α-SMA, but these cells did not appear to change in number or in their expression of α-SMA in response to FGF-2. FGF-2 has been shown to inhibit α-SMA in bone marrow–derived osteoprogenitors (40) but not in our mesenchyme-derived progenitor cell cultures from adult bone. Human cell cultures had approximately 20% α-SMA+ cells compared with 10% seen in mouse cell cultures, but no significant differences in α-SMA+ cells were found with age in either species. However, in the untreated human mesenchyme-derived progenitor cell cultures, a significant increase in α-SMA+ cells was found with time in culture, 24–72 hours, but not in the mouse cell cultures.

In conclusion, FGF-2 is able to stimulate the proliferation of osteoblast progenitors that express ALCAM in mesenchyme-derived progenitor cell cultures from aging mice and humans. FGF-2 appears to have therapeutic potential in stimulating the proliferation of osteoprogenitors that are decreased during aging and can be studied with mouse mesenchyme-derived progenitor cells with applicability to humans.

FUNDING

This work was supported by a grant from International Team for Implantology (ITI) Foundation, Basel, Switzerland, and National Institutes of Health grant AG021189.

Acknowledgments

The authors wish to acknowledge the technical assistance of Chris Desesa and Ankur Jhaveri.

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