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J Dent Res 86(1):79-83, 2007
© 2007 International and American Associations for Dental Research

RESEARCH REPORT
Biomaterials & Bioengineering

Effective Bone Engineering with Periosteum-derived Cells

H. Agata1,2, I. Asahina3,*, Y. Yamazaki4, M. Uchida1, Y. Shinohara1, M.J. Honda1, H. Kagami1, and M. Ueda1,2

1 Division of Stem Cell Engineering, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan;
2 Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan;
3 Department of Regenerative Oral Surgery, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan; and
4 Department of Plastic and Reconstructive Surgery, Kitasato University School of Medicine, Kanagawa, Japan

* corresponding author, 1-7-1 Sakamoto, Nagasaki 852-8588, Japan, asahina{at}nagasaki-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bone augmentation via tissue engineering has generated significant interest. We hypothesized that periosteum-derived cells could be used in place of bone marrow stromal cells (which are widely used) in bone engineering, but the differences in osteogenic potential between these 2 cell types are unclear. Here, we compared the osteogenic potential of these cells, and investigated the optimal osteoinductive conditions for periosteum-derived cells. Both cell types were induced, via bFGF and BMP-2, to differentiate into osteoblasts. Periosteal cells proliferated faster than marrow stromal cells, and osteogenic markers indicated that bone marrow stromal cells were more osteogenic than periosteal cells. However, pre-treatment with bFGF made periosteal cells more sensitive to BMP-2 and more osteogenic. Transplants of periosteal cells treated with BMP-2 after pre-treatment with bFGF formed more new bone than did marrow stromal cells. Analysis of these data suggests that combined treatment with bFGF and BMP-2 can make periosteum a highly useful source of bone regeneration.

KEY WORDS: marrow stromal cells • periosteal cells • osteogenic potential • bFGF • BMP-2


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bone defects that do not heal spontaneously need bone reconstruction for the recovery of bone function. While autografting is the gold standard in bone reconstructive surgery, autografts involve donor site morbidity. Artificial bone substitutes are also utilized; however, results are inconsistent, because these materials lack osteogenic potential. Recently, tissue engineering has attracted considerable attention (Young et al., 2005). Because it requires only a small amount of tissue from the patient, bone reconstruction by this technique is less invasive and is safer than conventional methods. Bone marrow stromal cells have multi-lineage differentiation potential and can therefore differentiate into cells with an osteogenic phenotype. Accordingly, they have been frequently used for bone reconstruction (Luria et al., 1987; Haynesworth et al., 1992; Matsubara et al., 2005). Periosteum-derived cells have also been used recently (Breitbart et al., 1998; Perka et al., 2000). There is a growing requirement for dentists to regenerate alveolar bone as a regenerative therapy for periodontitis and in implant dentistry. Concerning the donor site, it is easier for general dentists to harvest periosteum than marrow stromal cells, because they can access the mandibular periosteum during routine oral surgery. However, the differences in osteogenic potential between marrow stromal cells and periosteal cells remain unclear. In the present study, we compared the osteogenic potential of periosteum-derived cells and marrow stromal cells, after treatment with basic fibroblast growth factor (bFGF) and bone morphogenetic protein-2 (BMP-2), both of which have significant effects on osteogenesis (Rosen and Thies, 1992; Marie, 2003). Recently, Fakhry et al.(2005) showed that the combined use of bFGF and BMP-2 greatly enhances the osteogenic potential of chick embryonic calvaria-derived cells. Therefore, using BMP-2 and bFGF under the conditions described by Fakhry, with modifications, we attempted to determine the optimal osteo-inductive conditions for human mandibular periosteum-derived cells.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Cultures
The study conformed to the tenets of the Declaration of Helsinki, and the protocol was approved by the Ethical Committees of both Kitasato University and the Institute of Medical Science, The University of Tokyo. All subjects provided written informed consent.

Human periosteal tissue (1 cm2) was obtained from the mandibular angle of six patients (ages [yrs] 18, 19, 20, 21, 21, 24; gender, one male and five females) during the course of oral surgery. The excised sections of tissue were plated into 10-cm dishes (TPP, Zollstrasse, Trasadingen, Switzerland) containing {alpha}-MEM (Kohjin Bio, Saitama, Japan), supplemented with 10% fetal bovine serum (JRH Bioscience, Lenexa, KS, USA) and 1% penicillin-streptomycin-glutamine (serum-conditioned {alpha}-MEM, Invitrogen, Carlsbad, CA, USA), and were cultured at 37°C in 5% CO2. The medium was replaced every 2 days. When the cultures reached 90% confluence, cells were passaged and re-plated in 150-cm2 flasks (Corning, Big Flats, NY, USA).

Bone marrow stromal cells were obtained by iliac aspiration from three male volunteers (ages [yrs] 29, 33, 48), and were seeded in flasks and maintained in serum-conditioned {alpha}-MEM. The following day, floating cells were removed, and the medium was replaced with fresh medium. Passages were performed when cells reached 90% confluence. To ensure phenotypic uniformity, we used each cell lineage at the same passage (from 2 to 6) in the subsequent experiments.

Pre-treatment with Basic FGF
Cells were pre-treated with bFGF under conditions described elsewhere (Fakhry et al., 2005), with modifications. Both cell types were cultured in serum-conditioned {alpha}-MEM in dishes to 60% confluence. At that point, the serum-conditioned {alpha}-MEM was replaced with medium containing 1 ng/mL recombinant human bFGF (donated by Professor Y. Tabata, Kyoto University, Kyoto, Japan) and 100 {alpha}M ascorbic acid (Wako, Osaka, Japan), and the cells were cultured for 2 more days. Then, the cells were detached with trypsin-EDTA and re-plated (FGF pre-treatment).

Cell Proliferation Assay
On day 0, each cell type was plated at a density of 2.0 x 104 cells/mL/well in 12-well plates (Greinerbio-one, Kremsmuenster, Austria) containing serum-conditioned {alpha}-MEM. Cell numbers were counted directly in triplicate, and the medium was replaced with fresh serum-conditioned {alpha}-MEM on days 1 and 3.

Alkaline Phosphatase Activity Assay
On day 0, both cell types (with and without bFGF pre-treatment) were plated at a density of 1.0 x 105 cells/mL/well in 12-well plates containing serum-conditioned {alpha}-MEM. On day 1, either 1, 5, or 10 ng/mL bFGF or 30, 100, or 300 ng/mL recombinant human BMP-2 (donated by Astellas Pharma Inc., Tokyo, Japan) was added to each well, along with 100 µM ascorbic acid. On day 4, the medium was replaced with fresh medium containing identical growth factors. On day 7, cells were harvested and extracted with 20 mM HEPES (Dojindo, Kumamoto, Japan) buffer (pH 7.5) containing 1% Triton X-100 (Wako). Alkaline-phosphatase-specific (ALP) activity was assayed as described previously (Asahina et al., 1993). ALP activity is expressed as µmol p-nitrophenol/min/µg protein.

Reverse-transcription/Polymerase Chain-reaction
For RNA preparation, on day 0, both cell types (with and without FGF pre-treatment) were plated in 10-cm dishes containing serum-conditioned {alpha}-MEM. Cells were treated with or without 100 ng/mL BMP-2, 100 µM ascorbic acid for the following 6 days. Media were completely replaced on days 1 and 4. On day 7, total RNA was extracted with the use of TRIZOL reagent (Invitrogen). RNA samples (1 µg) were reverse-transcribed with Superscript III® reverse-transcriptase and oligo-dT primers (Invitrogen), according to the manufacturer’s protocol. For PCR amplification, we used primer sequences that have been described previously (Wordinger et al., 2002; Kamata et al., 2004). We analyzed gene expression of glyceraldehyde-3'-phosphate dehydrogenase (GAPDH), type I collagen (Col I), ALP, osteopontin (OP), osteocalcin (OC), BMP-2, BMP-4, and BMP receptors (BMPr) IA, IB, and II. After amplification, samples were analyzed by electrophoresis on a 1.5% agarose gel, and were visualized by ethidium bromide staining.

Transplantation of Cells into Immunodeficient Mice
For each transplantation, 1 x 106 harvested cells (with or without pre-treatment), in 1 mL of serum-conditioned {alpha}-MEM, were mixed with 50 mg of ß-tricalcium phosphate (ß-TCP) granules (Osferion®; Olympus, Tokyo, Japan) in a 14-mL polypropylene tube (Becton Dickinson, Franklin Lakes, NJ, USA). For the ensuing 6 days, cells were cultured in media with 100 ng/mL BMP-2 and 100 µM ascorbic acid, or without these additives as a control. The medium was replaced with fresh identical medium on day 4. After culture, each cell mixture was transplanted into a 6-week-old female BALB/cAJcl-nu/nu mouse (Nihoncrea, Tokyo, Japan). Five subcutaneous pockets were created in the back of each mouse, under anesthesia with diethyl ether, and the cell mixture was transplanted. As a negative control, ß-TCP alone was prepared and implanted into the mice. The transplants were harvested after 4 wks. NIH guidelines for the care and use of laboratory animals were observed in all procedures.

Histomorphometric Analysis of the Transplants
The harvested samples were fixed in 4% formaldehyde, decalcified, and embedded in paraffin wax. Then, 5-µm-thick sections were prepared from the middle of each transplant and stained with hematoxylin and eosin. The volume of newly formed bone was analyzed with Scion Image software (NIH, Bethesda, MD, USA), as described previously (Alsberg et al., 2001); the bone area is expressed as the percentage of total area (27.2 mm2).

Statistical Analysis
Results are expressed as mean values ± standard deviation. Statistical analysis of differences between groups was performed with Student’s t test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cell Proliferation
Proliferation of periosteal cells was significantly greater than that of bone marrow stromal cells (Fig. 1aGo). Although the number of periosteal cells was similar to that of marrow stromal cells on day 1, periosteal cells were twice as numerous as marrow stromal cells on day 3. Periosteal cells proliferated much faster than did marrow stromal cells, and had reached 100% confluence by day 6. Up to and including passage 6, all periosteal cells (regardless of donor) proliferated faster than the fastest-proliferating marrow stromal cells; in later passages, both cell types proliferated slightly slower (data not shown). FGF-pre-treated periosteal cells proliferated slightly slower than non-pre-treated periosteal cells (Fig. 1bGo), but they still proliferated faster than marrow stromal cells.


Figure 1
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  		Figure 1. Periosteal cells (closed circles) and marrow stromal cells (open circles) were plated in 12-well plates at a density of 2.0 x 104 cells/well. Cell numbers were counted directly (a). FGF-pre-treated periosteal cells are indicated by closed triangles, and non-pre-treated periosteal cells are indicated by closed circles (b). Values are means ± standard deviation for 3 cultures. Asterisk indicates significant difference in the number of periosteal cells, compared with marrow stromal cells on the same day; p < 0.05.

 
Alkaline Phosphatase Activity
Recombinant human bFGF had an inhibitory effect on ALP activity in both cell types, in a dose-dependent manner. Continuous exposure to bFGF (6 days) did not induce osteogenic differentiation in either cell type (Fig. 2aGo). Treatment with BMP-2 significantly enhanced the ALP activity of marrow stromal cells, but did not enhance the ALP activity of periosteal cells (Fig. 2bGo). FGF pre-treatment enhanced the responsiveness of periosteal cells to BMP-2; at doses of 100 and 300 ng/mL BMP-2, the ALP activity of FGF-pre-treated cells was about 3 times higher than that of the non-pre-treated cells (Fig. 2cGo). This effect of FGF pre-treatment on the BMP-2 induced increase in ALP activity was weaker for bone marrow cells than for periosteal cells (Fig. 2dGo).


Figure 2
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  		Figure 2. Periosteal cells (black) and marrow stromal cells (white) were plated in 12-well plates at a density of 1.0 x 105 cells/well. Then, the cells were incubated with bFGF (a) or BMP-2 (b) for 6 days. Alkaline phosphatase activity was analyzed on day 7 of culture. In (a), asterisk indicates significant difference (p < 0.05), compared with the control (–). FGF-pre-treated periosteal cells are indicated by black hatched bars; non-pre-treated periosteal cells are indicated by black bars; FGF-pre-treated marrow stromal cells are indicated by white hatched bars; non-pre-treated marrow stromal cells are indicated by white bars (c,d). Values are means ± standard deviation for 3 cultures. In b–d, asterisk indicates significant difference (p < 0.05) between paired conditions.

 
Reverse-transcription/Polymerase Chain-reaction Assay
Gene expression of periosteal cells and marrow stromal cells was examined by RT-PCR (Fig. 3Go). Control periosteal cells expressed only Col I and GAPDH. Treating periosteal cells with BMP-2 enhanced their expression of ALP, OP, BMP2, BMP4, BMPrIA, BMPrIB, and BMPrII. FGF pre-treatment of periosteal cells enhanced their expression of ALP, OP, BMP2, BMP4, BMPrIA, and BMPrIB. Combined FGF pre-treatment and BMP-2 treatment of periosteal cells enhanced their expression of ALP, BMP2, BMP4, BMPrIA, BMPrIB, and BMPrII.


Figure 3
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  		Figure 3. RT-PCR analysis of periosteal cells and marrow stromal cells. For 6 days, each cell type was cultured in serum-conditioned {alpha}-MEM alone (–) or in serum-conditioned {alpha}-MEM containing BMP-2 (BMP). To evaluate the effect of FGF pre-treatment, we pre-treated some cells with bFGF for 2 days (Pre-treatment), and treated some cultures with BMP after bFGF pre-treatment (Pre-treatment + BMP).

 
Control marrow stromal cells expressed Col I, GAPDH, ALP, OC, BMPrIA, and BMPrII. Treating marrow stromal cells with BMP-2 enhanced their expression of ALP, OP, BMPrIA, BMPrIB, and BMPrII. FGF pre-treatment of marrow stromal cells enhanced their expression of OP, OC, BMP2, BMPrIA, and BMPrII, but it inhibited their expression of ALP. Combined FGF pre-treatment and BMP-2 treatment of marrow stromal cells enhanced their expression of ALP, OP, OC, BMP2, BMPrIA, and BMPrII.

Transplantation of Periosteal Cells
Control periosteal cells had formed little new bone at 4 wks after transplantation (Fig. 4aGo). Implantation of ß-TCP alone did not cause formation of any new bone (data not shown). Periosteal cells treated with BMP-2 alone or FGF pre-treatment alone also formed new bone (Figs. 4b, 4cGo); however, the amount of newly formed bone was less than that of periosteal cells treated with BMP-2 after FGF pre-treatment. Periosteal cells treated with BMP-2 after FGF pre-treatment had formed significant ectopic new bone at 4 wks after transplantation (Fig. 4dGo).


Figure 4
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  		Figure 4. Hematoxylin and eosin staining demonstrating in vivo bone formation. Transplants of untreated control periosteal cells (-) formed little bone (a). Transplants of periosteal cells that received BMP-2 treatment alone (BMP) (b) or received FGF pre-treatment alone (pre-treatment) (c) also formed new bone. Periosteal cells that were treated with BMP-2 after FGF pre-treatment (pre-treatment + BMP) formed significant amounts of new bone (d). Bar indicates 300 µm. Arrow indicates new bone. The difference in new bone volume between periosteal cells (black) and marrow stromal cells (white) was determined by histomorphometric analysis (e). Asterisk indicates significant difference (p < 0.05) between paired conditions. Values are means ± standard deviation for 3 sections of each sample.

 
Histomorphometric analysis showed that marrow stromal cells formed slightly more new bone than did periosteal cells under control conditions (untreated), after treatment with BMP-2 alone, and after FGF pre-treatment alone; however, these differences were not statistically significant. Periosteal cells treated with BMP-2 after FGF pre-treatment generated significantly more newly formed bone than did marrow stromal cells treated with BMP-2 after FGF pre-treatment. Periosteal cells treated with BMP-2 after FGF pre-treatment formed 3 times as much new bone as did periosteal cells treated with BMP-2 without FGF pre-treatment (Fig. 4fGo).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we compared the osteogenic potential and clinical usefulness of periosteal cells with those of marrow stromal cells.

The proliferation rate of periosteal cells was much greater than that of marrow stromal cells (Fig. 1aGo). Reports indicate that primary cultures of human bone cells obtained from different donors can have very different proliferation rates (Im et al., 2004). We observed differences in proliferation rate among the present donors, but all of the present periosteal cells proliferated faster than did the present marrow stromal cells. Also in the present study, both cell types retained high expansion potential at later passages (Sakaguchi et al., 2005). Thus, the use of periosteal cells may shorten the cell culture period, thereby reducing both cost and the risk of contamination.

In the present study, we used periosteal cells obtained from young adults. Aging reportedly affects the mitogenic activity of osteoprogenitor cells (Tanaka et al., 1999). There is a need for studies comparing the bone-forming ability of periosteal cells between old and young donors.

Many in vitro studies have indicated that bFGF has a mitogenic effect on osteoprogenitor cells (Tanaka et al., 1999; Shimoaka et al., 2002). In contrast, the present transient bFGF treatment inhibited the proliferation of periosteal cells, although the effect was not statistically significant (Fig. 1bGo). The discrepancy between these studies may be due to the method of FGF treatment. In previous studies, the bFGF treatment was of longer duration than in the present study. Cells committed to the osteoblast lineage have been found to have lower proliferative potential than uncommitted cells (Malaval et al., 1999). Although the present continuous treatment with bFGF inhibited ALP activity (Fig. 2aGo), consistent with results from a previous study (Kalajzic et al., 2003), cessation of bFGF treatment may induce osteoblastic differentiation of periosteal cells. This hypothesis is consistent with the present data regarding osteoblastic gene expression of cells pre-treated with FGF. FGF pre-treatment increased the expression of molecules considered early-stage markers of osteogenic differentiation, such as ALP (Fig. 3Go) (Ryoo et al., 2006).

BMP-2 and bFGF have been found to induce osteogenic differentiation (Canalis et al., 1988; Yamaguchi et al., 1991), but, in the present study, the response to BMP-2 was greater for marrow stromal cells than for periosteal cells. Control periosteal cells showed less ALP activity and expressed fewer osteogenic markers than control marrow stromal cells. This suggests that marrow stromal cells are further differentiated along the osteoblastic cell lineage than are periosteal cells. Previous studies indicated that transient treatment with FGF, followed by treatment with BMP-2, enhanced osteogenic differentiation in vitro (Hanada et al., 1997; Fakhry et al., 2005). Similarly, in the present study, FGF pre-treatment enhanced responsiveness to BMP-2, and this enhancement was much stronger in periosteal cells than in marrow stromal cells. This suggests that the enhancement of osteogenic potential after FGF pre-treatment was due to an increase in the number of cells in the pre-osteoblast or osteoblast lineage.

Consistent with previous in vitro studies, the present combination of FGF pre-treatment and BMP-2 treatment enhanced the bone-forming potential of periosteal cells, which formed a greater volume of new bone than did marrow stromal cells in vivo. Thus, periosteal cells cultured under conditions that promote osteogenesis may be as useful for bone tissue engineering as are marrow stromal cells, and offer the advantage of greater proliferation.


   ACKNOWLEDGMENTS
 
This study was supported by a Grant-in-aid for Scientific Research (No. 16659547) from the Japan Society for the Promotion of Science, and by Grants-in-aid from Hitachi Medical Corporation (Japan) and Denix International Corporation (Japan).

Received February 21, 2006; Last revision September 8, 2006; Accepted September 26, 2006


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 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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