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J Dent Res 83(1): 27-34, 2004
© 2004 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Mesenchymal Stem Cells Acquire Characteristics of Cells in the Periodontal Ligament in vitro

P.R. Kramer1,*, S. Nares2, S.F. Kramer1, D. Grogan1, and M. Kaiser1

1 Department of Biomedical Sciences, Baylor College of Dentistry, Texas A&M University System Health Science Center, 3302 Gaston Ave., Dallas, TX 75246; and
2 Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, Bethesda, MD 20892;

* corresponding author, Pkramer{at}tambcd.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mesenchymal stem cells differentiate into multiple types of cells derived from mesenchyme. Periodontal ligament cells are primarily derived from mesenchyme; thus, we expected mesenchymal stem cells to differentiate into periodontal ligament. Using a combination of immunohistochemistry and in situ hybridization on co-cultures of mesenchymal stem cells and periodontal ligament, we observed a significant increase in mesenchymal stem cells’ expression of osteocalcin and osteopontin and a significant decrease in expression of bone sialoprotein, characteristics of periodontal ligament in vivo. Increased osteopontin and osteocalcin and decreased bone sialoprotein expression was detected within 7 days and maintained through 21 days of co-culture. We conclude that contact or factors from periodontal ligament induced mesenchymal stem cells to obtain periodontal-ligament-like characteristics. Importantly, analysis of the data suggests the feasibility of utilizing mesenchymal stem cells in clinical applications for repairing and/or regenerating periodontal tissue.

KEY WORDS: mesenchymal stem cell • periodontal ligament • regeneration • repair


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Repair of periodontal ligament appears to involve progenitor cells, present in the periodontal region, capable of forming fibroblasts, osteoblasts, and cementoblasts (Melcher, 1976). These cells appear clustered near blood vessels in the periodontal ligament and have characteristics of early progenitor cells (Gould et al., 1977, 1980; McCulloch, 1985). Moreover, progenitor cells in endosteal spaces potentially migrate from the bone to periodontal ligament to form tissues of the periodontium (McCulloch et al., 1987). Currently, it is unknown if a single progenitor cell type can give rise to all the tissues of the periodontium, but the presence of periodontal ligament and residing progenitor cells is necessary to prevent ankylosis upon re-implantation of extracted teeth (Boyko et al., 1981; Nyman et al., 1985; Van Dijk et al., 1991). Interestingly, paravascular localization of periodontal ligament progenitor cells suggests that blood or bone marrow could be the source of these cells (Gould et al., 1977;McCulloch et al., 1987). Repair and/or regeneration of bony defects by auto- or allografts produces some gain in attachment levels and has value in inducing periodontal ligament and cementum (Cortellini and Bowers, 1995; Reynolds and Bowers, 1996), but because grafts can become encased in fibrous connective tissue and junctional epithelium, many cases show little osteoinductive capacity (Dragoo and Sullivan, 1973; Moskow et al., 1979). Repair by guided tissue generation protocols to prevent apical gingival migration is clinically popular but problematic because of the variable results, limited clinical improvement, and susceptibility to infection (Tempro and Nalbandian, 1993; Pontoriero and Lindhe, 1995; Bratthall et al., 1998). Thus, there is a need for more predictable clinical methods.

Mesenchymal stem cells have the potential to produce different types of oral tissues and be utilized clinically to repair or regenerate oral damage. An understanding of the mechanisms and genes by which stem cell differentiation occurs would lead to more efficacious clinical treatments. Mesenchymal stem cells, precursors of mesenchymal tissues, can be derived from bone marrow stroma and produce cells such as chondrocytes, osteoblasts, adipocytes, and myoblasts (Pereira et al., 1995; Pittenger et al., 1999; Jiang et al., 2002). A single bone-marrow-derived cell was found to give rise to multiple tissue types (i.e., lung, gut, skin epithelia) (Krause et al., 2001). As a result, investigators analyzed the potency of bone-marrow-derived mesenchymal stem cells, which led to the additional conclusion that such cells can form neuroectodermal tissues, displaying neuronal and astrocytic markers after transplantation but showing rudimentary cellular morphology (Akiyama et al., 2002; Zhao et al., 2002). Moreover, total bone marrow samples give rise to neurons and astrocytes indistiguishable from the native cells (Brazelton et al., 2000), suggesting that an undisclosed pluripotent cell is yet present within the bone marrow (Kopen et al., 1999; Mezey et al., 2000). Importantly, rejection of allogenic stem cells by an immunocompetent host is negligible after implantation, probably due to the immunosuppressive effects of these cells (Uchida et al., 1998; Bartholomew et al., 2002; Di Nicola et al., 2002). Lack of an immune response suggests a high probability of success for the use of mesenchymal stem cells in clinical protocols. The most recent finding in this area supports and extends this concept by showing that tissue engineering with mesenchymal stem cells has been successful in producing bone (Boo et al., 2002; Partridge et al., 2002).

In our experimental paradigm, mesenchymal stem cells (from male donors) were tagged by fluorescent in situ hybridization (FISH) and then stained for a series of proteins that identify cell types present in the periodontium. Changes in mesenchymal stem cell morphology and protein expression were observed in these Y-chromosome-tagged cells (i.e., mesenchymal stem cells) to determine if they differentiated into a specific cell type (e.g., periodontal ligament, from female donors).


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Organotypic Tooth Explant Isolation and Culture
Samples of oral tissue were obtained for clinically necessary extractions with consent of the female human subjects following NIH guidelines and IRB approval. Extracted teeth were rinsed 3X in phosphate-buffered saline (PBS) containing penicillin and streptomycin and then once in minimal essential medium. The extracted tooth was held in place by a clamp on the crown of the tooth, the apical side facing up. Using a low-speed handpiece and a diamond disk, irrigated with 1X PBS, we removed a section from the extracted tooth at a depth of ~ 0.5 mm (Fig. 1Go, panels A1 and A2). The section was placed in minimal essential medium supplemented with 10% fetal calf serum and antibiotics and placed in 5% CO2 atmosphere (Okamoto et al., 1997). Proliferative cells having various morphologies were produced from the section in culture between 7 and 10 days. Cells with periodontal ligament morphology were diluted and cultured to confluence, then plated on glass slides and processed for immunohistochemistry after 24 or more hrs in culture. Human male mesenchymal stem cells were obtained from BioWhittaker Cell Biology Products (Walkersville, MD, USA) and cultured according to the manufacturer’s directions in MSCBM medium provided by the company. Undifferentiated cells are guaranteed between 2 and 4 passages. Criteria for undifferentiated mesenchymal stem cells are that the cells must be negative for blood cell or hematopoietic progenitor cell markers CD14, CD34, and CD45, plus express markers indicative of mesenchymal stem cells CD105, CD166, CD29, and CD44 (Pittenger et al., 1999; Le Blanc et al., 2003) and have the ability to differentiate into osteogenic, chondrogenic, and adipogenic lineages by means of specific growth medium (BioWhittaker Cell Biology Products). Mesenchymal stem cells were plated on glass slides or mixed with periodontal cells isolated from the organotypic tooth explants at ratios of 1:1, 2:1, and 10:1. Individual cell types and co-cultures were then placed in minimal essential medium supplemented with 10% fetal calf serum and antibiotics. Note: Altering the ratio of the 2 cell types did not have any observable effect on the differentiation events. Co-cultures of cells were isolated after 0, 3, 7, 14, and 21 days and processed for immunohistochemistry and in situ hybridization. Experiments were repeated at least three times with at least two different donors with similar results.



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  		Figure 1. Isolation and characterization of periodontal ligament. (A1) Cartoon depiction of a parasagittal cross-section through the jaw and attached tooth. Tooth slices (rectangle) were made in the apical region of the extracted tooth to avoid any contamination with gingival epithelial cells. Tooth slices, termed ‘organotypic tooth explants’, had dimensions of a half-millimeter depth, a width of 1–4 mm, and a height of 2–5 mm; variation was due to topography of the tooth and differences in circumference. (A2) Paraffin section (12 µm) from an organotypic tooth explant stained with hematoxylin and eosin. Organotypic tooth explants always consisted of cementum (C) and periodontal ligament (PDL) and dentin (D). In approximately 15% of the sections, a layer of alveolar bone (B) was present. (B1) Proliferative periodontal cells were then cultured in vitro from organotypic tooth explants. Greater than 80% of the cells cultured have a spindle shape resembling the morphological properties noted for human periodontal ligament cells (black arrows). (B2) Cultured cells from explants stain positive for collagen III (green fluorescence). Thus, periodontal ligament fibroblasts were isolated from the cellular population based on morphology and high levels of collagen III expression. (C1-G3) Staining of periodontal tissues for collagen III, osteopontin, osteocalcin, BMB-2/4, and bone sialoprotein in vivo was completed on 12-µm paraffin or fresh-frozen sections. Collagen III staining (brown, panel C2), osteopontin staining (brown, panel D2), osteocalcin staining (brown, arrows, panel E2), BMP-2/4 staining (brown, panels F1 and F2), and bone sialoprotein staining (brown, arrows, panels G2 and G3) of acute organotypic tooth explants. Panels C1, D1, E1, and G1 are controls where non-immune serum was added, instead of primary antibody, during the immunohistochemical procedure. In panels C1-G2, the bone (b), periodontal ligament (pdl), cementum (c), and dentin (d) tissue layers are indicated. Bars = 50 µm (B2, C1, C2, D1, G1, D2), 100 µm (E1, E2, G2), and 200 µm (F1). Panels F2 and G3 are high-magnification images of regions in panels F1 and G2, respectively.

 
Immunohistochemistry
Frozen or paraffin sections (20 µm) from tooth explants or cells, cultured on slides, were fixed (4% formaldehyde in 1X PBS) for 30 min. Explants were decalcified in 0.5 M EDTA (pH 6.8) for more than 10 days before being sectioned. The sections were blocked (10% normal goat serum or 5% bovine serum albumin in 1X PBS) and incubated in primary antibody > 12 hrs at 4°C. Primary antibodies used for staining included a goat polyclonal antibody for BMP-2/4 (R&D Systems, Minneapolis, MN, USA) diluted 1:20, rabbit polyclonal osteocalcin (Peninsula Labs, San Carlos, CA, USA) 1:500, monoclonal antibodies specific for collagen III (Chemicon, Temecula, CA, USA) 1:50, osteopontin (MPIIIB 101, Developmental Studies Hybridoma Bank, Iowa City, IA, USA) 1:5, and bone sialoprotein (WV1D1-9C5, Developmental Studies Hybridoma Bank) 1:5. Slides were then incubated with biotinylated goat anti-rabbit or goat anti-mouse 1:500 (Vector Labs, Burlingame, CA, USA) or rabbit anti-goat horseradish peroxidase (Biomeda, Foster City, CA, USA). Biotinylated secondary incubations were followed by streptavadin-horseradish peroxidase (Vectastain ABC kit, Vector Labs). Detection of a signal was completed by use of the DAB or AEC kit following the manufacturer’s directions (Vector Labs), and cells were counter-stained with hematoxylin and mounted. Bright-field images were captured with a DC290 digital camera (Eastman Kodak, Rochester, NY, USA).

Double-immunohistochemistry and in situ Hybridization
Fixed cells were processed for immunohistochemistry as described above, except that signal detection was completed with use of the TSA-Biotin tyramide signal amplification kit following the manufacturer’s directions (Perkin-Elmer Life Sciences Inc., Wellesley, MA, USA). Note: The primary antibody dilutions were increased by a factor of 2 in fluorescent labeling procedures (e.g., 1:5 becomes 1:10). Following the signal detection step, the slides were incubated in proteinase K (20 µg/mL in PBS and 0.1% SDS) at 37°C for 30–60 sec, rinsed in 1X PBS, and fixed for 10 min, followed by 3 rinses in 1X PBS, 5 min each. A Y-chromosome-specific probe (Mezey et al., 2000), random-prime-labeled with digoxigenin-11-dUTP following the manufacturer’s directions (Roche Diagnostics, Indianapolis, IN, USA), was mixed with hybridization buffer (50% formamide, 10% dextran sulfate, 0.02 M Tris, pH 7.4, 0.4 M NaCl, 1X Denhardt’s solution). The slides were coated, coverslipped, sealed with rubber cement, and heated to 95°C for 5 min (http://intramural.nimh.nih.gov/lcmr/snge/Protocols/ychrom.html). After an overnight incubation at 55°C, the slides were rinsed in 1X PBS, incubated with anti-DIG-POD (Roche Diagnostics), rinsed, and reacted with the TSA-Plus Cyanine 3 System (Perkin-Elmer) according to the manufacturer’s directions. Following the reaction, the slides were rinsed in 1X PBS, incubated with streptavidin SA-488 (Molecular Probes, Eugene, OR, USA), and mounted with DAPI stain. Controls included cells processed for immunohistochemistry using 10% normal goat serum in 1X PBS without addition of a primary antibody or without reacting with tyramide or processing for ISHH without the addition of a Y chromosome probe. Fluorescent images were captured with a CCD camera model 782-Y (Princeton Instruments, Princeton, NJ, USA). Images for staining localized within the cell were captured with an upright Leica TCS-P2 confocal microscope equipped with argon, krypton, and helium neon lasers with excitation wavelengths of 488, 543, and 633 µm, respectively. Leica confocal software was used to compile a series of images made through the interior of the cells, thus providing a cross-sectioned image.

Quantitation of Immunostaining
Fluorescent SA-488 staining of various proteins in the different cultures was captured on the CCD camera and quantitated by means of MetaMorph software (Universal Imaging Corporation, West Chester, PA, USA). For each experiment, all slides were processed simultaneously for a specific antibody, so that homogeneity in the staining procedure would be ensured between samples. After capture of the images at the same magnification, the threshold was set and maintained for each slide in the experiment, and the optical density was calculated by use of the morphometric analysis function within the software package. The optical density values from separate experiments with mesenchymal cells from two different donors and periodontal ligament cells from at least two different donors were combined. Optical density values were given as the mean ± SEM. Statistical significance was determined by one-way ANOVA, and comparison of different cultures was completed by means of the Mann-Whitney test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Periodontal Ligament
Immunohistochemical staining of sections made from organotypic tooth explants (Fig. 1Go, A1 and A2) indicated that adult periodontal ligament, cultured from the explants (Fig. 1Go, B1), expressed collagen III (Fig. 1Go, B2). Moreover, these cells had a spindle or spindle-like morphology (Fig. 1Go, B1). Collagen III (Fig. 1Go, C2), osteopontin (Fig. 1Go, D2), osteocalcin (Fig. 1Go, E2), and BMP-2/4 (Fig. 1Go, F1 and F2) were also expressed in explants taken acutely, thus representing in vivo expression. Collagen III staining was restricted to the periodontal ligament and was not detected in the cementum and dentin layers (Fig. 1Go, C2). Osteopontin staining was present in the periodontal ligament, and low-intensity staining was observed in the cementum (Fig. 1Go, D2). Osteocalcin staining was heterogeneous in the periodontal ligament (arrows), and staining was observed in the bone, dentin, and cementum (Fig. 1Go, E2). BMP-2/4 could be detected in the periodontal ligament, cementum, and bone tissue (Fig. 1Go, F1 and F2). Bone sialoprotein was present in the cementum, dentin, and alveolar bone (Fig. 1Go, G2 and G3). In contrast, periodontal ligament did not stain for bone sialoprotein (Fig. 1Go, G2 and G3). Non-immune serum controls showed no staining (Fig. 1Go, C1, D1, E1, and G1). Thus, cellular morphology—plus a pattern of high collagen III expression, the presence of osteopontin and osteocalcin, and the absence of bone sialoprotein—indicates that periodontal ligament can be differentiated from other periodontal tissues within the explant (i.e., dentin, cementum, and alveolar bone).

Mesenchymal Stem Cells Acquire Periodontal Ligament Characteristics
The staining pattern for mesenchymal stem cells was different from that seen for periodontal ligament. Mesenchymal stem cells have a low level of osteocalcin staining in comparison with periodontal ligament and show a flattened, cuboidal, or circular morphology at low-culture dilutions (compare Fig. 2Go, panels A and B, with panels C and D, respectively). Co-culture of periodontal ligament with mesenchymal stem cells for 7 days led to an overall change in the mesenchymal stem cell structure, to a more fibroblast-like morphology (Fig. 2EGo, arrows). Moreover, osteocalcin expression was significantly up-regulated in mesenchymal stem cells following co-culture for 7 and 21 days (Fig. 2IGo). Osteocalcin staining was localized to the interior of the cells, indicating that this protein was not bound to the outside membrane at detectable levels (Fig. 2HGo, arrows).



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  		Figure 2. Osteocalcin staining and Y-chromosome tagging of human female periodontal ligament and male mesenchymal stem cells. In panels A, C, E, and H, osteocalcin was fluorescently stained with SA-488 (green), and male mesenchymal stem cells were labeled for the Y chromosome (red dots). In panels B and D, osteocalcin staining was completed with DAB (brown). Osteocalcin staining of mesenchymal stem cells is shown in panel A (green) and panel B (brown). Periodontal ligament cells express high levels of osteocalcin in vitro, as shown in panel C (green) and panel D (brown). (E) Co-cultures of mesenchymal stem cells and periodontal ligament (1:1) after 7 days in vitro. Images from panels A, C, and E were from slides run in the same staining experiment and processed through the same solutions. Negative controls included: (F) periodontal ligament cells not treated with osteocalcin antibody (i.e., non-immune serum) and stained; and (G) periodontal ligament cells incubated with antibody but not reacted with biotin-tyramide (reactant necessary for streptavidin SA-488 binding and the fluorescent green staining). (H) Cross-section confocal image of the interior (arrows) of cocultured cells after osteocalcin staining (green). Horizontal and vertical lines indicate the X and Y planes through which the cross-sectioned images were made. Note: Mesenchymal cell is tagged by a red dot. (I) Histogram showing the mean optical density values for various cell groups after fluorescent osteocalcin staining, including mesenchymal stem cells (MSC), periodontal ligament cells (PDL), and co-cultures of MSC/PDL after 7 or 21 days. Optical density values were given as the mean ± SEM. Values were significantly different (P < 0.05), as shown by ANOVA. Asterisk indicates that the OD for MSC staining was significantly less (P < 0.05) than for the other 3 cell groups. N = 3 for MSC and MSC/PDL (7 days), and N = 10 for PDL and MSC/PDL (21 days). Bar = 50 µm (A, C, and E). Panels B, D, F, G, and H have the same magnification as panel A.

 
Mesenchymal stem cells express bone sialoprotein at high levels, but little bone sialoprotein was detected in the periodontal ligament (compare Fig. 3Go, panels A and B, with panels C and D, respectively). Co-cultures after 7 and 21 days showed that bone sialoprotein expression was reduced (Fig. 3Go, E and F). Expression of bone sialoprotein in mesenchymal stem cells was significantly reduced after co-culture for 7 days and continued to be reduced for at least 21 days (Fig. 3HGo). Control samples stained negative (Fig. 3GGo).



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  		Figure 3. Bone sialoprotein staining of periodontal ligament and male mesenchymal stem cells. In panels A, C, E, and F, cell cultures were stained with bone sialoprotein (green) and labeled for the Y chromosome (red dots). In panels B and D, bone sialoprotein staining was completed using DAB (brown). Bone sialoprotein staining of mesenchymal stem cells is shown in panel A (green) and panel B (brown). Periodontal ligament cells express low levels of bone sialoprotein in vitro, as shown in panel C (green) and panel D (brown). (E) Co-cultures of mesenchymal stem cells and periodontal ligament (1:1) after 7 days in vitro. (F) Mesenchymal stem cells (white arrows) express bone sialoprotein after co-culture (1:1) with periodontal ligament for 21 days. Panels A, C, E, and F are images taken from slides run in the same staining experiment and processed through the same solutions. (G) Negative control showing the DAB staining of periodontal ligament cells incubated with non-immune serum without bone sialoprotein antibody. (H) Histogram showing the mean optical density for mesenchymal stem cells (MSC) and periodontal ligament cells (PDL) and co-cultures after bone sialoprotein fluorescent staining. Optical density values were given as the mean ± SEM. Values were significantly different (P < 0.0001) as shown by ANOVA. Asterisk = P < 0.05, and double asterisk = P < 0.01 as compared with MSC. N >= 6. Bars = 50 µm (A, B, F) and 100 µm (C, D, E).

 
Mesenchymal stem cells express lower levels of osteopontin than periodontal ligament (compare Fig. 4Go, panels A and B, with panels C and D, respectively), and osteopontin staining in mesenchymal stem cells increased by 7 days of co-culture and continued to 21 days (Fig. 4Go, panels E and F, respectively). Quantitation of the osteopontin staining indicated that this increase was significant (Fig. 4HGo). Control samples stained negative (Fig. 4GGo). Bone sialoprotein and osteopontin were localized to the interior of the mesenchymal stem cells in co-cultures similar to osteocalcin (data not shown). Although periodontal ligament expressed BMP-2/4 and collagen III in vitro, we did not observe differences in staining compared with mesenchymal stem cells (data not shown).



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  		Figure 4. Osteopontin staining of periodontal ligament and male mesenchymal stem cells. In panels A, C, E, and F, cell cultures were stained with osteopontin (green) and labeled for the Y chromosome (red dots). In panels B and D, osteopontin staining was completed with the use of DAB (brown). Osteopontin staining of mesenchymal stem cells is shown in panel A (green) and panel B (brown). Periodontal ligament cells express high levels of osteopontin in vitro, as shown in panel C (green) and panel D (brown). In vitro co-cultures of mesenchymal stem cells and periodontal ligament (1:1) were incubated for 7 days (panel E) and 21 days (panel F). Periodontal ligament, mesenchymal stem cell, and co-culture images were from slides run in the same staining experiment and processed through the same solutions. (G) Negative control shows the DAB staining of periodontal ligament cells incubated with non-immune serum without osteopontin antibody. (H) Histogram showing the mean optical density for mesenchymal stem cells (MSC), periodontal ligament cells (PDL), and co-cultures after osteopontin fluorescent staining. Optical density values were given as the mean ± SEM. Values were significantly different (P < 0.0001), as shown by ANOVA. Double asterisk indicates that the OD for MSC staining was significantly less (P < 0.01) than that of the other 3 cell groups. N <= 6. Bars = 50 µm (F) and 100 µm (C, D, E). Panel C is the same magnification as A, and panel D is the same magnification as B.

 
Mesenchymal stem cells did not obtain the periodontal marker expression due to the culture conditions, since culturing the mesenchymal stem cells in BioWhittaker’s MSCBM medium or the minimal essential medium used for co-culturing did not change the OD of the 3 markers analyzed [osteocalcin (MSC medium, 249 ± 21; MEM medium, 200 ± 24); bone sialoprotein (MSC medium, 426 ± 20; MEM medium, 490 ± 41); and osteopontin (MSC medium, 81 ± 11; MEM medium, 78 ± 14)].


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
These studies tested the capacity of mesenchymal stem cells to differentiate into periodontal ligament cells in vitro. Periodontal ligament cells were identified through morphological characteristics and protein markers: collagen III, osteopontin, BMP-2/4, osteocalcin, and bone sialoprotein. The patterns of expression for these markers were similar in vitro and in vivo, with the exception of detectable amounts of bone sialoprotein found in cultured periodontal ligament. Co-culturing bone-marrow-derived mesenchymal stem cells and periodontal ligament led to a significant increase in mesenchymal stem cell expression of osteocalcin and osteopontin and a significant decrease in bone sialoprotein, characteristics of periodontal ligament in vivo. Increased protein expression was observed for osteopontin, osteocalcin, and bone sialoprotein after 7 days and continued for at least 21 days in culture. We conclude that contact or factors from periodontal ligament induced mesenchymal stem cells to obtain periodontal-ligament-like characteristics. Importantly, analysis of the data suggests the feasibility of utilizing mesenchymal stem cells in clinical applications for repairing and/or regenerating periodontal ligament tissue.

Protein Markers in Periodontal Ligament
The staining pattern we observed for collagen III, osteopontin, osteocalcin, and bone sialoprotein in the periodontal region in vivo was consistent with patterns reported previously (Lekic et al., 1996; Ivanovski et al., 2001). Moreover, our results indicate that the in vivo staining pattern of the periodontal ligament was similar to the cell staining pattern when cultured in vitro. BMP-2/4 and collagen III were expressed at similar levels (data not shown) in both mesenchymal stem cells and periodontal ligament and were not used as markers to differentiate between the two cell types. The BMP-2/4 staining pattern was inconsistent with that reported in previous studies, possibly due to the antibody specificity (Ivanovski et al., 2001). During root development, the pattern of staining for these proteins is likely different, suggesting that regenerative and repair processes are different (D’Errico et al., 1997), supporting the idea that care is required in obtaining donor explant tissue so that selection is from individuals of similar age (e.g., completed oral development in contrast to pediatric tissue).

Differentiation of Mesenchymal Stem Cells into Periodontal Ligament Fibroblasts
Significant increases in the optical density of osteocalcin and osteopontin staining and significant decreases in bone sialoprotein staining in mesenchymal stem cells after culture with periodontal ligament fibroblasts for 7 days were detected with little or no observable increase after longer periods. This observation was specific to the effects of co-culturing mesenchymal stem cells with periodontal ligament, since growth of the mesenchymal stem cells in BioWhittaker’s MSCBM medium or minimal essential medium did not cause a change in marker expression. Moreover, the change in marker expression was not due to excretion of a particular protein from the periodontal ligament and subsequent attachment to the membrane of mesenchymal stem cells, since confocal microscopy showed that the proteins were localized within the cell. Thus, analysis of the data, collectively, suggests that a contact-mediated factor(s) and/or secreted factor(s) induces the process by which mesenchymal stem cells obtain periodontal-ligament-like marker expression.

Collagen III and bone sialoprotein are useful markers in that they aid in differentiating among adult periodontal ligament (high collagen III, low bone sialoprotein expression), gingiva (low collagen III, low bone sialoprotein expression), cementum, dentin, and bone (absent or low collagen III, modest bone sialoprotein expression) (Lekic et al., 1996; Ivanovski et al., 2001). Moreover, osteocalcin and osteopontin are observed in the periodontal ligament, cementum, dentin, and bone, but little or none is observed in the gingiva (data not shown) (Ivanovski et al., 2001). These 4 proteins can form the basis for the identification of mesenchymal stem cell differentiation into various periodontal cell types. For example, if mesenchymal stem cells (modest collagen III, high bone sialoprotein expression) differentiated into gingival fibroblasts, we would observe a decrease in both markers. Thus, observed changes in gene expression of these 4 markers or other potential genes (i.e., cementum attachment protein, dentin sialoprotein) would determine the extent to which mesenchymal stem cells differentiate into the cell types present in the periodontal region.

In conclusion, our results demonstrate mesenchymal stem cells’ potency to develop periodontal ligament characteristics and suggest that the cells may have the potential to form other periodontal tissues. Moreover, we describe an in vitro periodontal model in which the genes or molecular pathways leading to these differentiation events can be determined. Studies of mesenchymal stem cells’ potential to form all the various periodontal ligament tissue types in vitro and in vivo, plus an understanding of this process, can yield highly beneficial clinical applications for repairing and/or regenerating periodontal tissue.


   ACKNOWLEDGMENTS
 
These studies were supported by funds from the Baylor Oral Health Foundation.

Received February 7, 2003; Last revision September 15, 2003; Accepted September 29, 2003


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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