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Keywords:

  • bone marrow stromal cells;
  • alveolar bone;
  • osteogenesis;
  • mesenchymal stem cells

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

We isolated and expanded BMSCs from human alveolar/jaw bone at a high success rate (70%). These cells had potent osteogenic potential in vitro and in vivo, although their chondrogenic and adipogenic potential was less than that of iliac cells.

Introduction: Human bone marrow stromal cells (BMSCs) have osteogenic, chondrogenic, and adipogenic potential, but marrow aspiration from iliac crest is an invasive procedure. Alveolar BMSCs may be more useful for regenerative medicine, because the marrow can be aspirated from alveolar bone with minimal pain.

Materials and Methods: In this study, alveolar bone marrow samples were obtained from 41 patients, 6–66 years of age, during the course of oral surgery. BMSCs were seeded and maintained in culture with 10% FBS and basic fibroblast growth factor. In addition, BMSCs were induced to differentiate into osteoblasts, chondrocytes, or adipocytes in appropriate medium.

Results and Conclusion: From a small volume (0.1–3 ml) of aspirates, alveolar BMSCs expanded at a success ratio of 29/41 (70%). The success rate decreased with increasing donor age, perhaps because of age-dependent decreases in the number and proliferative capacity of BMSCs. The expanded BMSCs differentiated into osteoblasts under osteogenic conditions in 21–28 days: the mRNA levels of osteocalcin, osteopontin, and bone sialoprotein, along with the calcium level, in alveolar BMSC cultures were similar to those in iliac cultures. However, unlike iliac BMSC, alveolar BMSC showed poor chondrogenic or adipogenic potential, and similar differences were observed between canine alveolar and iliac BMSCs. Subsequently, human alveolar BMSCs attached to β-tricalcium phosphate were transplanted into immunodeficient mice. In transplants, new bone formed with osteoblasts and osteocytes that expressed human vimentin, human osteocalcin, and human GAPDH. These findings suggest that BMSCs have distinctive features depending on their in vivo location and that alveolar BMSCs will be useful in cell therapy for bone diseases.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Bone marrow stromal cells (BMSCs) can differentiate into a variety of tissues—bone, cartilage, tendon, muscle, adipose tissue, and neuronal tissue—and their transplantation promotes regeneration of various tissues.(1–4) BMSCs have been isolated from various bones, including the ilium, femur, tibia, and spine,(5–8) but whether their proliferative and differentiation potentials depend on their in vivo location is unknown. Furthermore, marrow aspiration from these bones is an invasive procedure. Considering these facts, we decided to try collecting BMSCs from alveolar bone during the course of dental surgery, because most young adults undergo wisdom tooth extraction. We examined whether BMSCs could be expanded ex vivo from a small volume of alveolar bone marrow aspirates, and we also examined the effects of age, sex, disease history, and the volume of aspirates obtained from patients on ex vivo expansion of alveolar BMSCs. Furthermore, we compared the proliferative and differentiation potentials of alveolar BMSCs with those of iliac BMSCs, using human and canine marrow aspirates.

Alveolar BMSCs were cultured with basic fibroblast growth factor (bFGF), because BMSCs maintained with bFGF retained their differentiation potentials throughout many mitotic divisions.(5,6) The use of bFGF allowed us to expand alveolar BMSCs from a small volume (0.1-3 ml) of marrow aspirates, although alveolar BMSCs were not obtained from all alveolar aspirates of patients >50 years of age. Although the alveolar BMSCs showed a proliferative capacity and a potent osteogenic potential, the cells—unlike iliac BMSCs—had a poor adipogenic or chondrogenic potential, suggesting that in vivo location of BMSCs modulates their differentiation potentials.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Cell cultures

Human bone marrow was obtained from the alveolar bone or the ilium according to a protocol approved by ethical authorities at Hiroshima University. In this study, we selected patients whose bone marrow sites were opened during oral surgery to obtain marrow aspirates using routine syringes and needles without contamination by periodontal tissues. In extraction of impacted wisdom teeth or extirpation of cysts, alveolar bone around a tooth or a cyst was removed, and the bone marrow was exposed. Subsequently, the aspirate was obtained from the marrow site using an 18G injection needle (JMS, Hiroshima, Japan) connected to a disposable syringe (JMS). In cases of dental implant, the aspirate was obtained from drill holes in the alveolar bone. In cases of jaw deformities, the aspirate was obtained from the osteotomy groove along the anterior border of the mandibular ramus. In cases of mandibular fracture, the aspirate was obtained from the marrow site that was exposed by widening the gap between the fractured margins or from burr holes through which interosseous wire was passed. The aspirates were obtained from alveolar and/or jaw bones, but in this study, we refer to the BMSCs as alveolar BMSCs. In addition, alveolar and iliac bone marrow was obtained from 5- to 6-month-old female beagle dogs using a Komiya's puncture16G needle (1.5 × 25 mm; Kurita Injection Syringes, Tokyo, Japan). The puncture was made near lower molars under anesthesization. In pilot studies, a medium containing heparin was added to the marrow aspirates a few minutes after aspiration. However, this impaired ex vivo expansion of BMSCs, perhaps because of partial coagulation. Thus, in this study, marrow aspirates were mixed immediately with 1–3 ml of DMEM (Sigma), supplemented with 200 units/ml heparin. The cells were centrifuged at 500g for 5 minutes and resuspended with DMEM without heparin. Bone marrow cells including erythrocytes were seeded at a density of 0.1 ml aspirate/35-mm tissue culture dish (Corning) and maintained in 2 ml of DMEM supplemented with 10% FBS (Hyclone) and antibiotics (100 units/ml penicillin G and 100 μg/ml streptomycin; medium-A). Three days after seeding, floating cells were removed, and the medium was replaced by fresh medium-A. Thereafter, attached cells were fed with fresh medium-A supplemented with 1 ng/ml of bFGF, which was added every other day. Passages were performed when cells were approaching confluence: the cells were seeded at a density of 5 × 103 cells/cm2 on 60- or 100-mm tissue culture dishes (Corning) and maintained in 4 or 10 ml of medium-A supplemented with 1 ng/ml of bFGF.

Differentiation potentials of BMSCs

Osteogenic, chondrogenic, and adipogenic conversion of BMSCs was determined according to the procedures reported by Pittenger et al. with some modifications.(9) For osteogenic differentiation, cells were seeded at 4 × 104 cells/16-mm well (2.3 × 104 cells/cm2) and maintained for 21–28 days in DMEM supplemented with 10% FBS, 10 mM β-glycerophosphate, 100 nM dexamethasone, and 50 μg/ml ascorbic acid-2-phosphate. For chondrogenic differentiation, cells were seeded at 2.5 × 105 cells/15-ml plastic centrifuge tube and maintained for 28 days in 0.5 ml of serum-free α-MEM (high glucose) supplemented with 6.25 μg/ml insulin, 6.25 μg/ml transferrin, 6.25 ng/ml selenite, 5.33 μg/ml linolate, 1.25 mg/ml bovine serum albumin, 10 ng/ml transforming growth factor-β3, 100 nM dexamethasone, and 50 μg/ml ascorbic acid-2-phosphate. The cultures were fed with 0.5 ml of the medium until 3 days after seeding. Thereafter, the cultures were fed with 1 ml of the medium every other day. Sections of these pellets were stained with toluidine blue on day 28. For adipogenic differentiation, cells were seeded at 2 × 105 cells/35-mm well (2.3 × 104 cells/cm2) and grown to confluence in medium-A. Adipogenic differentiation was induced by subjecting confluent monolayers to three rounds of adipogenic treatments. Each consisted of incubation with adipogenic induction medium (DMEM-high glucose, 10% FBS, 0.2 mM indomethacin, 1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methyl-xanthine, and 10 μg/ml insulin) for 72–96 h, followed by incubation with maintenance medium (DMEM-high glucose, 10% FBS, and 10 μg/ml insulin) for another 72–96 h.

Glycosaminoglycan, alkaline phosphatase activity, calcium, GAPDH activity, and DNA

The glycosaminoglycan (GAG) content was determined using a sulfated GAG assay kit (Biocolor).(10) Alkaline phosphatase (ALP) activity was determined by the method of Bessey et al.(11) The calcium content was determined by the method of Gitelman.(12) GAPDH activity was determined using a GAPDH activity assay kit (Hokudo, Sapporo, Japan).(13) DNA was determined using a fluorescent DNA quantification kit (Bio-Rad).

RT-PCR analysis of BMSC cultures

Total RNA was extracted from cultures using Isogen (Nippon Gene, Tokyo, Japan). The first-strand cDNA was synthesized from 1 μg of total RNA using the SUPERSCRIPT II RNase H reverse transcriptase (Life Technologies). Using the cDNA as a template, PCR was carried out under the following conditions: denaturation at 94°C for 30 s and primer extension at 65°C for 1.5 minutes in 30 cycles. Pairs of nucleotides, 5′-GTCAAGGCCGAGAATGGGAA-3′ and 5′-GCTTCACCACCTTCTTGATG-3′ for GAPDH (GenBank Accession no., M33197, 613 bp), 5′-CATTTTGGGAATGGCCTGTG-3′ and 5′-ATTGTCTCCTCCGCTGCTGC-3′ for bone sialoprotein (J05213, 565 bp), 5′-CTAGGCATCACCTGTGCCATACC-3′ and 5′-CAGTGACCAGTTCATCAGATTCATC-3′ for osteopontin (J04765, 331 bp), and 5′-CCACCGAGACACCATGAGAG-3′ and 5′-CCATAGGGCTGGGAGGTCAG-3′ for osteocalcin (X53698, 419 bp) were used as primers for RT-PCR. Obtained PCR products were separated on 1% agarose gels and stained with ethidium bromide.

FACS analysis

BMSCs at passages 3–4 were harvested with trypsin and EDTA, centrifuged at 1500g for 5 minutes, and resuspended at 5 × 106 cells/ml in PBS containing 3% FBS. Aliquots containing 105 cells were incubated with individual primary antibodies or control IgG for 30 minutes at room temperature. The cells were washed in PBS containing 3% FBS and incubated with a fluorescent conjugated secondary antibody for 30 minutes at room temperature. Samples were analyzed using a FACSCalibur cytometer (Becton Dickinson), and the data were analyzed using the CELLQUEST software (Becton Dickinson). The following monoclonal antibodies (mAbs) were used: fluorescein isothiocyanate (FITC)-conjugated or R-phycoerythrin (PE)-conjugated antibodies against CD13, CD14, CD29, CD34, CD44, CD49b, CD54, CD56, CD71, CD90, CD105, CD106, CD117, CD124, CD138, CD144, HLA-ABC, HLA-DR, mouse-IgG1, mouse-IgG2a, or mouse IgM (Immunotech Coulter Company); antibodies against CD123, CD140b, CD166, or mouse-IgG3 (Pharmingen); antibodies against Flk-1 (Santa Cruz Biotechnology); antibodies against MT-MMP-1 (Sigma); antibodies against STRO-1 (Genzyme); antibodies against RANKL (R & D Systems), and anti-rabbit-IgG (Chemicon International).

Transplantation of BMSCs into mice

The potential for cells to differentiate into osteoblasts after transplantation into immunodeficient mice was assessed as described.(14,15) Human alveolar BMSCs at passages 3–5 (1.5 × 106 cells) in 1.0 ml of DMEM were mixed with 40 mg β-tricalcium phosphate (β-TCP) powder (OSferion; Olympus Co., Tokyo, Japan). After incubation at 37°C for 90 minutes, the mixture was centrifuged at 1500 rpm for 1 minute, and the supernatant was discarded. The pellet of β-TCP powder with adherent cells was mixed with 15 μl of mouse fibrinogen (3.3 mg/ml solution in PBS) and mouse thrombin (25 U/ml in 2% CaCl2; both from Sigma) to form a fibrin clot. The fibrin clots transplanted into 5-week-old female CB-17 scid/scid (SCID; severe combined immunodeficiency) mice (Nihoncrea, Tokyo, Japan). After anesthetizing by intraperitoneal injection with 10% Nembutal (Dainihon Seiyaku Co., Osaka, Japan) in PBS, five skin incisions were made on the dorsal surface of each mouse, fibrin clots (five per mouse) were transplanted, and incisions were sutured. The transplants were harvested 8 weeks after transplantation.

Immunohistochemical and histomorphometrical analyses of transplanted sample

The transplants were fixed in 4% paraformaldehyde for 1 day, decalcified with 10% formic acid for 3 days, and embedded in paraffin. Four-micrometer-thick sections were prepared in the middle of the transplants, collected on poly-l-lysine-coated slides, and stained using H&E for histochemical examination. To examine human vimentin expression immunohistochemically, endogenous peroxidase was quenched by incubating with 1% H2O2 and methanol. The sections were incubated with anti-human vimentin mAb (Dako; 50-fold dilution with Dako Antibody Diluent) for 1 h and treated with Envision (Dako). Color reaction was developed with diaminobenzidine (Dako). To examine osteocalcin expression, the sections were incubated with antihuman osteocalcin mAb (200-fold dilution; Biomedical Technologies, Stoughton, MA, USA) for 1 h, and color was developed with DAKO LSAB +System, HRP (Dako). Bone formation area in three fields (random sampling, 0.41 mm2/field) in the middle sections—stained with H&E—of the transplants of alveolar BMSC (a9) plus β-TCP powder or β-TCP powder alone was captured by CCD camera (coolpix 4500; Nikon, Tokyo, Japan), and the bone area in the pictures was traced with Adobe Photoshop version 7.0 (Adobe Systems, San Jose, CA, USA); the bone area is expressed as the percentage of total area (0.41 mm2).

RNA extraction from transplanted samples

Total RNA was prepared from the transplants as described.(16) Briefly, extracted samples were snap-frozen in liquid nitrogen and pulverized for 1.5 minutes at 2000 rpm in a mixer mill. A 1-ml aliquot of TriReagent (Sigma) was added directly to the powdered samples and warmed to room temperature. Each sample was transferred to a 1.5-ml microcentrifuge tube and mixed by orbital rotation for 10 minutes at room temperature. After the addition of 0.2 ml of chloroform, samples were vortexed and allowed to sit at room temperature for 15 minutes, and then centrifuged for 20 minutes at 12,000 rpm. The upper aqueous phase was removed and mixed with an equal volume of 70% ethanol. Total RNA was isolated using RNeasy minicolumns and reagents (Qiagen). DNase I (Ambion) treatment was performed to remove genomic DNA from RNA samples according to the manufacturer's protocol.

PCR analysis of implanted samples

First-strand cDNA was synthesized by Omniscript reverse transcriptase (Qiagen; 2 μg total RNA/20-μl reaction volume) using oligo dT primer (Promega). PCR amplification was performed (1 μl cDNA solution/25-μl reaction volume) using oligonucleotide primers corresponding to cDNA sequences for human-specific GAPDH (GenBank Accession no. M33197, 347 bp; sense 5′-CACCAGGTGGTCTCCTCT-3′, antisense 5′-GTACATGACAAGGTGCGG-3′), human-specific osteocalcin (X53698, 315 bp; sense 5′-CATGAGAGCCCTCACA-3′, antisense 5′-AGAGCGACACCCTAGAC-3′), mouse-specific osteocalcin (X04142, 443 bp; sense 5′-AACAGACAAGTCCCACACAG-3′, antisense 5′-GCTGTGACATCCATACTTGC-3′), and human and mouse GAPDH (M33197 and M32599, 268 bp; sense 5′-CACCAGGTGGTCTCCTCT-3′, antisense 5′-GTACATGACAAGGTGCGG-3′) using Taq polymerase (Promega). PCR was performed at 94°C/(45 s), 56°C/(45 s), and 72°C/(60 s) for 35 cycles. After amplification, 10 μl of each reaction was analyzed by 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining.

Oligonucleotide primers corresponding to cDNA sequences for the mouse-specific osteocalcin were designed using mouse sequence (X04142). The site selected for mouse-specific osteocalcin has low (<60%) homology to human osteocalcin (X53698). Even if these primers could anneal to human cDNA, the PCR product size would be 419 or 339 bp, which differs from 443 bp of the mouse product. Furthermore, we confirmed that no PCR product was synthesized with these primers and human RNA samples containing osteocalcin mRNA. In the case of human-specific GAPDH (M33197), the antisense primer had no similar homology site to mouse GAPDH (M32599). Thus, the product amplified by these primers corresponds to human GAPDH.

Statistical analysis

Student's t-test was used.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Expansion of alveolar BMSCs in culture

We obtained alveolar bone marrow samples from 41 dental patients. BMSCs (fibroblast-like cells) in 29 samples adhered to the culture surface and proliferated in the presence of 10% FBS and bFGF in primary and secondary cultures. No BMSC expansion was observed with the other samples (Table 1). There was no sex difference regarding the expansion of alveolar BMSCs (Fig. 1A), but the BMSC expansion depended on patients' age (Fig. 1B): BMSCs from patients >50 years of age often showed only a few adherent cells in primary cultures, and these did not form colonies within 14 days (data not shown), suggesting an age-related decline in the number of alveolar BMSCs and/or their growth capability. Marrow samples obtained during the course of wisdom tooth extraction or surgery for bone fracture or jaw deformity showed a higher success ratio than did those obtained during surgery for dental implants or dental cyst extraction (Fig. 1C). It remains unknown whether the site of aspiration affects the success ratio (Table 1). The low success ratio for dental implants may be caused by the increased age of the patients (Table 1). Whereas the success ratio increased with an increase in the volume of obtained marrow samples (Fig. 1D), this relationship is questionable because some aspirates contain peripheral blood.

Table Table 1. Isolation of BMSCs From Alveolar Bone
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Figure Fig. 1.. Effects of sex, age, disease history, and marrow sample volumes on ex vivo expansion of alveolar BMSCs. Cells in marrow aspirates were seeded and maintained as described in the Materials and Methods section. *The number of patients examined.

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All BMSC lines obtained by ex vivo expansion were maintained at least until passages 3–4 and then stored in liquid nitrogen for differentiation assays; some lines underwent further successive passages for proliferation assays (Fig. 2). Passages were performed when cells were approaching confluence. The cell number was determined at the passage, and the cumulative cell number at the passage is shown in Fig. 2. Alveolar BMSC lines retained their proliferative capacity until passages 8–11. The growth rate during the logarithmic growth phase (1.0 ± 0.2 cell division/day; doubling time, 25 ± 4 h) and proliferative life span (36 ± 10 cell doublings) of alveolar BMSCs were similar to those (0.9 ± 0.1, 28 ± 5, and 33 ± 11, respectively) of iliac BMSCs, although both BMSC lines showed large interindividual variations (Fig. 2).

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Figure Fig. 2.. The growth and proliferative life span of alveolar and iliac BMSCs. Alveolar (a) and iliac BMSC lines (i) obtained from the secondary cultures were seeded and passaged in 10-cm tissue culture dishes at 5000 cells/cm2. The ages of donors (i1, i2, i3, i4, i5, a1, a2, a3, a4, a8, and a9) were 22, 24, 22, 32, 29, 19, 25, 29, 31, 24, and 19 years, respectively. Passages were performed when cells were approaching confluence. Each point shows the cumulative cell number at the passage. The number of passages is shown in parenthesis.

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Osteogenic differentiation of human alveolar BMSCs in vitro

In most cultures of alveolar BMSC lines (a1-a11) maintained in osteogenic conditions for 21–28 days, the mRNA levels of osteopontin, osteocalcin, bone sialoprotein, and ALP (Fig. 3A), as well as the calcification level estimated with alizarin red (Fig. 3B), were higher than those in undifferentiated cultures maintained in medium-A alone. The calcium level in a1-a11 cultures in osteogenic conditions was significantly higher than in undifferentiated cultures (Fig. 3C), but the degree of calcification varied among the lines. Lines a4, a5, and a7 showed the expression of osteopontin and bone sialoprotein at low levels and calcification at high levels on day 28. In contrast, lines a1, a6, and a9 showed expression of osteopontin and bone sialoprotein at moderate or high levels and calcification at low levels on day 28: The latter may represent a delayed calcification process.

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Figure Fig. 3.. The osteogenic potential of alveolar BMSCs. Alveolar BMSCs lines (a1-a11) obtained at passage 3 were induced to differentiate into osteoblasts by incubating with the osteogenic medium (OS+) for (B) 21 or (A and C) 28 days. In addition, line a5 was maintained in medium A (OS−) for (B) 21 or (A and C) 28 days. The expressions of osteopontin (OP), osteocalcin (OC), bone sialoprotein (BSP), and ALP mRNA in triplicate cultures was determined by RT-PCR on day 28. (B) Cultures maintained in the osteogenic medium (OS+) or in medium A (OS−) were stained with alizarin red on day 21. (C) The calcium level was determined on day 28. Values are means ± SD for four cultures. *p < 0.05.

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Poor chondrogenic and adipogenic potentials of alveolar BMSCs

The differentiation potential of alveolar BMSCs was compared with that of iliac BMSCs using a12-a14 and i1-i3 lines. In the osteogenic conditions, ALP activity and calcium level in alveolar BMSC cultures were similar to those in iliac cultures (Fig. 4A). The chondrogenic potential of alveolar and iliac BMSCs was examined in pellet cultures, because chondrocyte differentiation takes place at high levels in pellet cultures.(17,18) Almost all iliac cells reorganized into a cartilage-like tissue that was stained purple with toluidine blue (Fig. 4B), whereas scarcely any metachromasia was observed with alveolar BMSCs, indicating poor chondrogenesis in the alveolar cell pellets. In the periphery of alveolar cell pellets, a few chondrocytes appeared (Fig. 4D), but the GAG level and ALP activity were lower in alveolar cultures than in iliac cultures (Fig. 4B). Under the adipogenic conditions, most iliac BMSC prosperously accumulated lipid droplets, whereas only a few adipocytes appeared in cultures with alveolar BMSC (Figs. 4C and 4E). Furthermore, alveolar cells showed a lower GAPDH, a marker for adipocytes, than did iliac cells on day 28. Some alveolar BMSC lines showed appreciable adipogenic potential on day 45, and their adipogenic potential was greater than that of fibroblasts (data not shown).

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Figure Fig. 4.. The differentiation potentials of human alveolar and iliac BMSCs. (A) Human alveolar (a12-a14) and iliac BMSCs (i1-i3) obtained at passage 3 were transferred into the osteogenic medium. The cell layers were stained with alizarin red on day 21. ALP activity and the calcium content of the cell-matrix layers were determined on day 28. (B) Human alveolar and iliac BMSCs obtained at passages 3–4 were incubated in the chondrogenic status in pellet cultures for 28 days and stained with toluidine blue. The levels of GAG and ALP were determined on day 28. In the periphery of the alveolar BMSC pellet, stained with toluidine blue, a few chondrocytes were observed (D). (C) Human alveolar and iliac BMSCs obtained at passage 3 were maintained in the adipogenic status for 28 days. The cells were stained with oil red O. GAPDH activity was determined on day 28. Values are means ± SD for four cultures. *p < 0.05. (E) Magnification of the alveolar BMSC culture maintained in the adipogenic status for 28 days: a few cells became adipocytes in alveolar cultures.

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To compare alveolar BMSCs with iliac BMSCs without interindividual variations, we simultaneously isolated alveolar and iliac BMSCs from three beagle dogs. After osteogenic differentiation, ALP and calcium levels in the alveolar BMSC cultures were found to be similar to those in the iliac cultures (Fig. 5). However, under chondrogenic conditions, the alveolar BMSCs synthesized glycosaminoglycan or ALP, and these cells synthesized GAPDH under adipogenic conditions, but only marginally in both cases. In contrast, the iliac BMSCs underwent either chondrogenic or adipogenic differentiation under these conditions (Fig. 5).

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Figure Fig. 5.. The differentiation potentials of canine alveolar and iliac BMSCs. (A) Iliac and alveolar BMSCs were isolated from three dogs (d1-d3). Cells obtained from secondary cultures were incubated in the osteogenic status. The cell layers were stained with alizarin red on day 21. ALP activity and the calcium content of the cell-matrix layers were determined on day 28. (B) Iliac and alveolar BMSCs obtained from the secondary cultures were transferred into the chondrogenic medium in pellet cultures for 28 days and stained with toluidine blue. The levels of GAG and ALP activity were determined on day 28. (C) Alveolar and iliac BMSCs obtained from the secondary cultures were maintained in the adipogenic status for 28 days. The cells were stained with oil red O. GAPDH activity was determined. Values are means ± SD for four cultures. *p < 0.05; **p < 0.01.

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Cell surface antigens

Previous studies have shown that several cell surface antigens, including ALCAM (activated leukocyte cell adhesion molecule), CD29 (integrin β-1), intercellular adhesion molecule (ICAM)-1, platelet-derived growth factor receptor (PDGFR), CD44, CD90, and CD105/SH2, are expressed in BMSCs and/or perichondrium mesenchymal stem cells.(9,19,20) In this study, we examined the expression of 25 cell surface antigens in alveolar and iliac BMSCs by FACS analysis: None of the cell surface antigens examined differed between alveolar and iliac BMSCs (Fig. 6). STRO-1 is a marker for BMSCs.(21) However, alveolar and iliac BMSCs showed STRO-1 expression at a low level (Fig. 6). Previous studies have also shown that STRO-1+ cells in undifferentiated human BMSC populations are only 7 ± 6%.(22) Therefore, STRO-1 may be progressively lost with time in these cultures.

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Figure Fig. 6.. Cell surface antigen expression by human alveolar and iliac BMSCs. Human alveolar and iliac BMSCs obtained at passage 3 (maintained in OS− medium) reacted with antibodies to cell surface antigens and were analyzed using a FACSCalibur cytometer.

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Transplantation of alveolar BMSCs

Next we examined whether alveolar BMSCs could differentiate into bone tissue in vivo. Alveolar BMSCs were attached to β-TCP powder and transplanted into SCID mice: 8 weeks after transplantation, new bone formation was observed (Figs. 7B and 7C). In contrast, no bone formation was observed with implants of β-TCP alone (Figs. 7A). Histomorphometrical measurements showed significant differences in bone formation between cell-loaded and unloaded implants: in six transplants (18 fields) with alveolar BMSCs plus β-TCP, bone area was 22 ± 9% compared with 0 ± 0% in four implants (12 fields) with β-TCP alone (Table 2), proving that alveolar BMSCs do indeed enhance initial bone formation.

Table Table 2. Bone Area in the Middle Sections of Transplants With Alveolar BMSCs
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Figure Fig. 7.. In vivo differentiation of human alveolar BMSCs after transplantation. Three lines of alveolar BMSCs (a9, a10, a11) obtained at passages 3–5 were used. Implants of (A and D) β-TCP powder alone or (B, C, E, and F) β-TCP + BMSC (a9) were stained with (A-C) H&E or (D-F) human-specific anti-vimentin mAb. (G) RT-PCR analysis of transplants of three alveolar BMSC lines was performed for detection of human cells using human-specific GAPDH and osteocalcin primers and mouse cells using human and mouse GAPDH primers and mouse-specific osteocalcin primers. Bar = (A, B, D, and E) 200 and (C and F) 50 μm.

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It was still unknown, however, whether transplanted BMSCs contributed to the bone formation, because host (mouse) cells could have induced bone formation in response to β-TCP. To address this issue, we used mAb against human vimentin that did not cross-react with mouse vimentin. The mAb to human-specific vimentin reacted with osteoblasts and osteocytes around and in new bone tissues: most osteoblasts and osteocytes, as well as some mesenchymal cells surrounding bone tissue, in the transplants with BMSCs were stained with the human-specific antibody (Figs. 7E and 7F) compared with no human vimentin+ cells in the implants with β-TCP alone (Figs. 7D). Thus, human alveolar BMSCs did in fact reorganize into new bone tissues. Furthermore, osteoblasts, osteocytes, and mesenchymal cells in or near bone tissues in the transplants of alveolar BMSC synthesized osteocalcin (Fig. 8).

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Figure Fig. 8.. Immunohistochemical photographs of alveolar BMSC transplants with anti-osteocalcin antibody. Specimens were immunostained (B) with or (A) without primary antibody (anti-osteocalcin). Bar = 50 μm.

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To examine whether new bone formation would occur with other alveolar BMSC lines, we examined the expression of human osteocalcin and human GAPDH mRNA, using total RNA isolated from whole transplants. In almost all transplants (11/13) of three alveolar BMSC lines (3/3, 4/6, and 4/4), human osteocalcin and/or human GAPDH mRNA expressions were observed (Fig. 7G), and similar bone formation was observed with human iliac BMSCs (data not shown). In contrast, no human osteocalcin/GAPDH expression was detected in the implants of β-TCP alone, although mouse GAPDH was found in these implants. Unexpectedly, using mouse-specific osteocalcin primers, mouse osteocalcin mRNA was detected in some transplants of human alveolar BMSCs, suggesting that mouse mesenchymal cells partly contributed to bone formation during osteogenic differentiation of human BMSCs. However, no mouse osteocalcin mRNA was detected in the implants of β-TCP alone (Fig. 7G), indicating the absence of bone formation in whole implants of β-TCP alone. These findings indicate that in vivo bone formation in the transplants of alveolar BMSCs was much greater than that in the implant of β-TCP alone.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Alveolar BMSCs had the same fibroblastic shape as that reported for BMSCs isolated from the iliac crest, and their proliferative and osteogenic potentials were similar to those of iliac BMSCs. However, alveolar BMSCs hardly differentiated into chondrocytes, and their adipogenic potential was less than that of iliac BMSCs, at least in the standard differentiation medium: we do not know at present whether other growth factors not included in the medium might enhance the chondrogenic or adipogenic differentiation of these cells. However, we rarely observed cartilage callus formation at the site of a jaw fracture, which may be caused by the poor chondrogenic potential of alveolar BMSCs. The physiological significance of the low adipogenic potential of alveolar BMSCs is not known, but, in any case, we showed here for the first time that topologically different bone marrows contain BMSCs with different features.

Precisely why alveolar and iliac BMSCs have different features remains unknown. BMSCs, like hematopoietic stem cells,(23,24) might have migrated from some bones to others,(20) because it has been suggested that BMSCs migrate from bone marrow to injured tissues.(25,26) And BMSC-like cells with osteogenic, adipogenic, and chondrogenic potentials have, in fact, been found in the blood.(27) It is likely that the features of BMSCs are modulated by the components of the adjacent bone marrow, such as growth factors/cytokines and extracellular matrix: different bones may have different marrow components.

In this study, we examined the effects of age, sex, disease history, aspiration site, and volume of aspirate and found a significant correlation between age and success ratio for BMSC expansion. This is a revealing observation, because the decrease in skeletal bone formation and rate of fracture repair observed with aging is reportedly caused by a decrease in numbers of BMSCs and/or their osteogenic capacity or changes in their secretion of cytokines, such as interleukin-11 and insulin-like growth factor binding protein-3.(28,29) However, there have been conflicting reports on the effect of age on human BMSCs. Stenderup et al.(30) reported that there was no age-related difference in the number and proliferative capacity of human osteogenic cells derived from marrow aspirates, and Oreffo et al.(31) found no age-related difference in the colony-forming efficiency of BMSCs or in ALP+ colony-forming efficiency, although they did find a reduction in the size of BMSC colonies with age. On the other hand, Nishida et al.,(32) Mueller and Glowacki,(8) and D'Ippolito et al.(7) showed an age-related decline in the osteogenic potential of BMSCs from, respectively, human iliac crest, femur, and vertebral bone. In this study, we found an age-related, marked decline in the proliferative capacity of alveolar BMSCs. Alveolar BMSCs may be more affected by age than iliac BMSCs. In any case, considering the age-dependence of the proliferative potential of alveolar BMSCs, alveolar BMSCs isolated from young patients during wisdom tooth extraction could be preserved in liquid nitrogen at −196°C for future clinical use.

Transplantation of β-TCP plus alveolar BMSCs induced bone formation in the skin of SCID mice. Furthermore, the alveolar BMSC-derived osteoblasts and osteocytes synthesized osteocalcin adjacent to or in newly formed bone, and all bone tissues formed on the surface of β-TCP powder (Fig. 7 and data not shown), suggesting that the contact between BMSCs and the calcium phosphate scaffold enhanced osteogenic differentiation even in the nonskeletal tissue. Mouse mesenchymal stem cells also contributed to bone formation in the transplants of human BMSCs, perhaps because growth factors released by a large number of transplanted BMSCs enhanced osteogenic differentiation of a small number of endogenous mesenchymal cells in vivo. These findings suggest that alveolar BMSCs have a strong osteogenic potential in the presence of appropriate scaffolds and that the number of BMSCs/mesenchymal cells near scaffolds is important for bone formation in vivo.

We feel that the most important significance of this study is the introduction of alveolar BMSCs as a cell source for regenerative medicine, especially because alveolar BMSCs can be isolated with minimal pain to the patient. In addition, these cells have a shown osteogenic potential both in vitro and in vivo. Furthermore, their poor adipogenic potential may decrease unfavorable fat formation during tissue regeneration at the site of transplantation. Transplantation of bone marrow or BMSCs has been shown to promote bone formation in patients with osteogenesis imperfecta or long bone defect,(33,34) and we are now investigating whether transplantation of alveolar BMSCs can promote regeneration of alveolar bone in patients with periodontal disease.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

The authors thank dental doctors (T Ishikawa, T Okamoto, S Toratani, R Tani, and N Domen) and patients in the Department of Oral and Maxillofacial Surgery and Oral Medicine, Hiroshima University Hospital, for the isolation of bone marrow samples. This study was supported by the Japan Science and Technology Corporation.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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