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Laboratorio di Differenziamento Cellulare (I.M., A.M., G.C., R.C., R.Q.), Istituto Nazionale per la Ricerca sul Cancro/Centro di Biotecnologie Avanzate, 16132 Genova, Italy; Dipartimento di Informatica (I.M.), Sistemistica e Telematica (DIST), Università di Genova, 16145 Genova, Italy; and Dipartimento di Oncologia Clinica e Sperimentale (R.C.), Università di Genova, 16132 Genova, Italy
Address all correspondence and requests for reprints to: Rodolfo Quarto M.D., Laboratorio di Differenziamento Cellulare, Istituto Nazionale per la Ricerca sul Cancro/Centro di Biotecnologie Avanzate, Largo Rosanna Benzi no. 10, 16132 Genova, Italy.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A considerable amount of literature has been published on the therapeutic applications of cells of bone origin (3, 4, 5). Several laboratories have shown the potential of bone marrow stromal cells (BMSC) in bone reconstitution (6, 7, 8, 9). BMSC contain a multipotent self-renewing compartment and are capable of differentiating into several mesenchymal lineages: osteoblasts, chondrocytes, adipocytes, and myocytes (10). When implanted in vivo, these cells reconstitute bone and in some conditions cartilage (6, 8, 9, 11). In particular, when combined with mineral containing tridimensional scaffolds, they form a primary bone tissue highly vascularized and colonized by host hemopoietic marrow (8, 11). In this system, the ceramic provides a tridimensional structure, a cell adhesion site, and may act as a primer for the formation of new bone matrix foci; in addition, BMSC differentiate into osteoblasts and deposit extracellular matrix on the ceramic surface.
To be used for clinical therapy, the BMSC system needs to be highly standardized and reproducible in bone formation efficiency. Frequency of bone formation can vary depending on the different human bone marrow primary cultures and strongly depends on cell culture conditions (8). Bone formation was observed only when the cells were cultured in the presence of FBS from highly selected lots, making it necessary to perform laborious serum testing before its use (8).
The effects of growth factors on proliferation and activity of bone cells and marrow stromal cells have been investigated by several groups (12, 13, 14, 15, 16, 17, 18).
The possibility to increase BMSC in vitro proliferation rate and osteogenic potential has been investigated. We have studied the biological effects of different growth factors and hormones on ex vivo BMSC expansion, and here we report culture conditions that represent a significant improvement in the technology to expand ex vivo human BMSC maintaining their osteogenic potential.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human skin fibroblasts were obtained from surgical specimens. Samples were minced and plated. Skin fragments were held to the bottom of the culture dishes by sterile coverglasses until a significant cell growth was reached. Fibroblasts were expanded both with and without FGF-2 as described for BMSC.
Colony forming efficiency
To evaluate colony forming efficiency (expressed as ratio
between the number of colonies obtained and the number of cells
plated), 5 x 105 nucleated bone marrow cells were
plated in 60-mm dishes (
1.8 x 104
cells/cm2). The medium was changed after 4 days and then
twice a week. After 2 weeks, cells were washed with PBS, pH 7.2, fixed
with formaline 4% in PBS, stained with 1% methylene blue in borate
buffer (10 mM, pH 8.8) for 30 min and then washed with
distilled water. Images of the dishes were acquired by a Cohu black and
white camera, digitized by LG-3 frame grabber card (Scion Corporation,
Frederick, MD) into a Quadra 840AV Macintosh computer and analyzed with
the NIH-Image public domain software, developed at the U.S. National
Institutes of Health, and available on the Internet at
http://rsb.info.nih.gov/nih-image/. On this platform, a particular
computer program (Macro) was integrated to automatically process each
dish image: colonies were counted, their total area was calculated, and
the average number and size of colonies formed in different culture
conditions was derived.
Growth curve
At day 0, 5 x 103 first passage cells were
plated in each well of a 24-well plate; cell number was then evaluated
at different days via Thiazolyl blue (MTT) staining (19). Briefly,
culture medium was removed and replaced with 0.5 ml of medium without
FCS; then 25 µl MTT (Sigma, St. Louis, MO) stock solution (5 mg/ml)
were added to each culture being assayed. After 3-h incubation, the
medium was removed and the converted dye solubilized with acidic
isopropanol (0.04 N HCl in absolute isopropanol).
Absorbance of converted dye was measured at a wavelength of 570 nm with
background subtraction at 670 nm. Conversion to cell number was based
on a standard curve.
Alkaline phosphatase (AP) activity
AP activity was determined as the rate of conversion of
p-nitrophenyl phosphate to p-nitrophenol. For each condition to be
tested, 2.5 x 104 cells were plated in four 10-mm
wells; two wells were washed with PBS and 100 µl 0.01% SDS were
added; after 10 min, a prewarmed solution of 0.5 ml substrate (Sigma
catalog no. 104–100) and 0.5 ml alkaline buffer solution (Sigma
catalog no. 221) were added in each of the two wells. After a 15-min
incubation at 37 C, the contents of each well were added to 10 ml 0.05
N NaOH to quench the reaction and absorbance was read at
410 nm. The remaining two wells were used to perform the MTT assay to
normalize AP activity to cell number.
Cells were stained for AP using kit no. 85L-3R (Sigma) and following the direction of the manufacturer. To evaluate the number of AP-positive BMSC colonies, dishes were stained for AP and positive colonies counted. The dishes were then counterstained with hematoxylin, total number of colonies counted, and percentage of AP-positive derived. To evaluate the percentage of AP-positive BMSC expanded in different conditions, at least 300 cells were counted in each of the three independent experiments and percentage of AP-positive BMSC was derived.
In vitro deposition of mineralized matrix
BMSC cultures were expanded both with and without FGF-2. After
reaching confluence, cultures were continued for 2 weeks in the absence
of FGF-2 and stimulated in medium supplemented with 10% FCS with the
addition of 50 µg/ml ascorbic acid (Sigma), 150 µg/ml sodium
ßGlycerophosphate (Sigma), and 10-8 M
dexamethasone (Sigma) every other day. After 2 weeks, cells were fixed
with formaline 4% in PBS, extensively rinsed in distilled water,
stained for 10 min with 2% Alizarin S (pH 4.1), and rinsed twice with
absolute ethanol. Quantification of mineralized matrix was performed by
computer-assisted analysis on digitized images of the samples. Basal
level of mineral deposition was derived from unstimulated cultures and
used as threshold to binarize images and to measure the percentage of
positive area. Duplicate dishes were processed for MTT assay to
normalize data on cell number.
Ectopic bone formation and analysis of the bone volume
The material selected for in vivo implantation was a
highly porous ceramic support based on 100% hydroxyapatite (HA), of
70–80% porosity. Pore size distribution was: <10 µm, 3% vol;
10–150 µm, 11% vol; >150 µm, 86% vol. The material was
produced and kindly provided by Fin-Ceramica Faenza (Faenza, Italy) and
dry sterilized for 4 h at 200 C. As control material, in some
experiments collagen were used sponges (produced and kindly provided by
Coletica, Lyon, France).
In these experiments, only cells from adult donors were used. In our protocol, after 3–4 weeks expansion (corresponding to the first passage), stromal cells were detached from the dishes with 0.05% trypsin and 0.01% EDTA, washed in serum-free medium, and resuspended at 2.5 x 105 cells/20 µl. Two 10-µl aliquots were then applied on two opposite faces of dry ceramic cubes (approximate volume, 64 mm3), so that each block was loaded with 2.5 x 105 stromal cells. The loading volume was determined on the basis of the average volume of fluid blocks could adsorb. Ceramic blocks were sc implanted just after loading.
When fresh bone marrows were used, nucleated cells were isolated by Ficoll gradient centrifugation, counted, and each cube was loaded with the appropriate number of cells resuspended in two aliquots of 10 µl serum-free medium.
Recipient nude mice (CD-1 nu/nu) of 1 month of age were purchased from Charles River Italia (Calco, Italy), kept in a controlled environment, and given free access to food and water. Mice were cared for and treated according to institutional guidelines. Animals were anesthetized by intramuscular injection of xilazine (1 mg/50 ml) and ketamine (3 mg/50 ml). Grafts were implanted sc on the back of the mice (up to six implants for each animal). Animals were killed 4 and 8 weeks after implantation, grafts removed, and processed for histological analysis. Samples were decalcified (Osteodec, Bio-Optica, Italy) according to manufacturer’s instructions, paraffin embedded, sectioned, stained with hematoxylin/eosin, and analyzed for bone tissue using a Zeiss Axiophot microscope (Oberkochen, Germany) and the NIH-Image image analysis software on a Quadra 840AV Macintosh Computer. For each sample, two different depths were analyzed, two histological sections were prepared for each level, and four to six images for each section were acquired to evaluate the amount of bone formation. Bone tissue quantitation was therefore performed for a total area of approximately 70 mm2 per each sample. The amount of bone formed was assessed as percent of bone vs. total tissue, thus excluding the volume occupied by HA.
To determine whether newly formed bone within the ceramic cubes was derived from donor human cells or host mouse cells, tissue sections were stained with the biotinylated human specific BC-2 antitenascin monoclonal antibody (kindly provided by Dr. L. Zardi, Genova, Italy) (20). Mouse bone sections were used as control. Immunoperoxidase staining was performed on paraffin embedded samples as described in Manduca et al. (21).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To account for the variability in colony number and size observed in
cultures derived from marrows obtained from different donors, in each
experiment values of the different conditions were normalized to the
ones obtained with 10% FCS alone. After 2 weeks in 10% FCS, the
average colony number was 20.8 ± 5.5, and the average colony size
was 3.2 ± 0.8 mm2. Colony number was not affected by
most of the factors tested (P > 0.05), whereas the
addition of FGF-2 resulted in a colony count of 14.6 ± 2.0,
corresponding to 30 ± 0.1% reduction (P < 0.01)
(Table 1
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). The other factors did not
influence significantly BMSC colony size (Table 1
).
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Colonies obtained with the addition of the different factors were
analyzed for AP expression (Table 1). AP-positive colonies increased in
cultures treated with EGF and Dex (1.38 and 2.08-fold, respectively)
and decreased in cultures treated with FGF-2 and TGF-ß1 (0.65 and
0.46-fold, respectively).
Because FGF-2 was the most active in increasing colony size and was significantly decreasing the percentage of AP positive colonies, we concentrated on its effects on BMSC in vitro.
Morphological differences were rather evident in primary cultures
between control cells and BMSC cultured with FGF-2 (Fig. 2, a and b). In fact, cells cultured with
this factor were thinner and more elongated (b). In the following
passages, the original fibroblast-like phenotype was gradually lost by
the control cultures which assumed a more flattened cell phenotype
(Fig. 2c), whereas it was maintained when FGF-2 was present in the
culture medium (Fig. 2d).
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). At confluence, average cell density
was at least 2-fold higher in the cultures treated with FGF-2.
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In vitro deposition of calcified matrix was investigated.
After BMSC expansion to confluence both with and without FGF-2,
cultures were stimulated for 2 weeks with ascorbic acid,
ßGlycerophosphate and dexamethasone, with no further FGF-2 addition.
Dishes were stained with Alizarin S to detect calcium salt deposition,
as described in the Materials and Methods section. BMSC
expanded both with and without FGF-2 were also cultured for the same
time without any treatment besides the normal change of medium and used
to derive basal levels of Alizarin staining. After 2 weeks of treatment
with ascorbic acid, ßGlycerophosphate, and dexamethasone, calcium
deposition was detected in both BMSC populations, and it was higher
when cells were previously expanded with FGF-2. Results were quantified
by computer-assisted image analysis (Fig. 4). Mineral deposition was found to be
significantly higher in FGF-2 expanded BMSC (4.3- and 2.7-fold higher,
respectively, before and after normalization for cell number) (Table 2). Comparable results were obtained with
von Kossa staining (data not shown).
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). At 8 weeks, bone formation reached
12.66% of the total tissue in the case of FGF-2 expanded BMSC and
4.4% in the case of Dex expanded BMSC, it remained below 0.5% in the
other conditions tested (Table 3).
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. Implants without
attached cells were used as controls, and they showed formation of a
vascularized loose connective tissue but no evidence of newly deposited
bone (Fig. 5a). As an additional control, we implanted HA cubes loaded
with human skin fibroblasts cultured with FGF-2; in these samples, we
did not detect any bone (data not shown). In our assay, implants with
cells expanded in 10% FCS showed little bone matrix deposition
(<0.5% of total formed tissue) (Fig. 5b). Newly formed bone was
present in nearly all the samples loaded with cells treated with FGF-2
(Fig. 5c); at higher magnification (Fig. 5d), osteoblasts were observed
at the edge of the bone tissue, facing mesenchymal, undifferentiated
cells. Bone matrix was abundant and osteocytes were enclosed in
lacunae. The amount of matrix deposited, quantified using image
analysis techniques, varied depending on the primary culture but
averaged 12.66 ± 2.05% of the total formed tissue (Table 3).
From this percentage, considering the average implant volume (64
mm3) and the average ceramic porosity (75%), we calculated
a total bone formation in 8 weeks of approximately 6.1 mm3
per implant starting from 2.5 x 105
FGF-2 expanded BMSC.
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FGF-2 expanded BMSC were also loaded onto collagen sponges and implanted sc in nude mice. Samples were harvested 8 weeks after implantation and processed for histological analysis. These samples displayed only a loose connective tissue but neither bone nor cartilage.
Osteogenic potential of freshly harvested whole bone marrows was also
investigated. Samples of freshly harvested bone marrows were adsorbed
onto ceramic cubes and sc implanted in nude mice as described in the
Materials and Methods section. When 5 million nucleated
cells (1 ml fresh bone marrow) were implanted, the amount and
characteristics of bone tissue formed were similar to those observed in
the experiments where 250,000 expanded BMSC were used. In a similar
experiment, 250,000 nucleated cells from fresh bone marrow did not
induce any detectable bone formation.
To confirm the origin of the cells within the bone in the ceramic
cubes, tissue sections were immunostained with the biotinylated human
specific BC-2 monoclonal antibody against tenascin, no counterstaining
was performed. The antibody clearly reacted with osteocytes,
osteoblasts, and undifferentiated mesenchymal cells of the newly formed
bone tissue (Fig. 6B) but did not stain
mouse bone cells (Fig. 6C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In vitro expansion and tissue-specific delivery of autologous BMSC would certainly represent an important advance in skeletal tissue repair. Several reports have shown the therapeutic potential of BMSC in bone reconstitution (3, 4, 5). However, to be used for cellular therapy, the BMSC system needs to be highly standardized and reproducible for bone formation efficiency. In fact, frequency of bone formation can vary among different human bone marrow primary cultures and strongly depends on cell culture conditions. In the attempt to standardize culture conditions, we have studied the biological effects of different growth factors and hormones on ex vivo expansion of BMSC.
Our data suggest that EGF and FGF-2 are the most active in increasing colony size, i.e. growth rate, whereas Dex and FGF-2 are the most active in promoting bone formation. Being the most active in promoting both BMSC growth rate and bone formation, FGF-2 has been the only factor selected for further studies. The apparent absence of effect of the other factors (PDGF, GH, IGF, etc.) tested may be explained by the presence in the serum used of the same factors in already maximally stimulatory concentrations. An alternative explanation may be that BMSC secrete these factors in an autocrine fashion.
FGF-2 is a potent modulator of proliferation and activity of bone cells (12) and marrow stromal cells (13, 14, 15, 16, 17, 18), although it might not always act directly (13). In our experiments, we noted that the addition of FGF-2 to the BMSC culture medium in the presence of serum yielded a reproducible and constant level of cell proliferation and in vivo bone reconstitution. In fact, 83% (5 out of 6) of the primary cultures treated with FGF-2 yielded quantitatively higher amounts of bone as compared with control cultures, where only 1 out of 6 promoted the deposition of detectable bone matrix.
FGF-2 expanded BMSC maintained unaltered their original elongated phenotype for a longer time. This finding may be explained either by a direct effect of FGF-2 on differentiation or commitment level of these cells, or by FGF-2 stimulation of the synthesis and/or organization of extracellular matrix components, which would induce an alteration in the spreading and consequently in the cell phenotype. Interestingly, FGF-2 expanded BMSC expressed lower levels of AP activity (although percentage of AP-positive cells was similar to the one found in BMSC expanded in FCS alone) and displayed in vitro a higher osteogenic differentiation potential. These data support the idea that FGF-2 is able to support growth and expansion of osteogenic precursors. Not all factors (i.e. EGF) promoting proliferation of the total BMSC population do also maintain the osteogenicity of the expanded cells, thus suggesting that FGF-2 is not only involved in stimulation of BMSC proliferation but also in the maintenance of a particular functional state. It has been described that FGF-2 can substitute for the apical ectodermal ridge to maintain cells in a responsive state required to allow patterning of developing limb (26). It would be interesting to speculate that, in our system, FGF-2 is required to maintain cells in a stem state. Further studies at the clonal level are in progress to understand whether in the presence of FGF-2 certain CFU-f are stimulated or others are inhibited and to determine whether the selected cell population still retains mesenchymal stem potential.
It has been reported that BMSC differentiate in vitro in several mesenchymal lineages (10); however, in vivo only cartilage and bone formation has been described (6, 8, 11). In our experiments, because we have used an open system (i.e. available for vascular ingrowth), we observed only bone tissue. Interestingly, bone matrix deposition was always polarized with the newly formed bone facing HA ceramic and the osteoblasts located at the boundary between the newly formed bone and the mesenchymal undifferentiated cells. Bone was never observed when BMSC were implanted on a collagen sponge. We suggest that the mineralized surface of the ceramic may serve as a primer for the initiation of bone matrix deposition.
In agreement with other authors (8, 27), using a monoclonal antibody specific for human tenascin, we have shown that in our implants bone formation was of donor origin.
We also compared the osteogenic potential of BMSC expanded in vitro in the presence of FGF-2 vs. freshly harvested bone marrow to estimate the osteogenic power of the cells expanded in our conditions. Considering the expansion factor of the cultured BMSC, their osteogenic potential was highly increased with respect to the starting bone marrow sample. This fact per se makes it possible to conceive autogenic bone reconstruction in patients without the need for a large bone marrow aspirate, which is always inconvenient and sometimes impossible. We did observe a decrease in the osteogenic efficiency of the expanded BMSC with respect to the fresh bone marrow, but this finding was not unexpected because it is conceivable that during the expansion some of the dividing cells remain in the stem/progenitor compartment, whereas others may start to progress down different lineage pathways. Alternatively, the cotransplanted hemopoietic cells, virtually absent in the cultured stromal cell population, may create an optimal microenvironment (in terms of growth factors and cytokines secreted) for the stromal cells to proliferate and differentiate.
In the present paper, we have described culture conditions that represent a significant improvement of the standard techniques for a rapid and reproducible expansion of osteogenic precursors from human bone marrow. In particular, we have shown the role of FGF-2 in maintaining osteogenic potential of BMSC during their ex vivo expansion.
The association of these cells with the appropriate bioceramics is a model for a potentiated guided regeneration of bone, where ceramic tridimensional scaffold provides the track for tissue regeneration, allows blood vessel invasion, and primes multicentric bone formation by BMSC. Such a biomaterial might be considered both osteoconductive and osteoinductive and possibly be used in human therapy as a high efficiency technique for large bone segment reconstruction.
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Acknowledgments |
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Footnotes |
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2 Recipient of a fellowship from Fondazione Anna Villa Rusconi
(Varese, Italy).
Received March 7, 1997.
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0.05) in clonogenic growth
occurred only in response to EGF and FGF-2.
