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J Dent Res 81(3): 203-208, 2002
© 2002 International and American Associations for Dental Research

RESEARCH REPORT
Biological

{alpha}-Smooth-muscle Actin in and Contraction of Porcine Dental Pulp Cells

D.P. Brock1, R. Marty-Roix2, and M. Spector2,*

1 Harvard School of Dental Medicine, Boston, MA; and
2 Department of Orthopaedic Surgery, MRB 106, Brigham and Women's Hospital, 75 Francis Street, Harvard Medical School, Boston, MA 02115;

*corresponding author, mspector{at}rics.bwh.harvard.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The finding of expression of a muscle actin isoform, {alpha}-smooth-muscle actin (SMA), by the stromal cells of several tissues prompted this study of SMA expression by cells derived from the porcine dental pulp. The SMA content of the cells increased with time in culture. These SMA-containing cells were found to have the capability to contract a collagen-glycosaminoglycan analog of extracellular matrix in vitro.

KEY WORDS: smooth-muscle actin • pulp • contraction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The cellular population of the dental pulp cells includes fibroblasts, odontoblasts, cells associated with the neurovascular supply, and, to a lesser degree, cells of the immune system (Berkovitz et al., 1992). It has been suggested that the predominating pulpal fibroblast-like stromal cell has the capability to change its phenotype, reflecting different morphologies and functions (Berkovitz et al., 1992). Stromal cells adjacent to the odontoblastic region can be induced to differentiate into dentin-secreting odontoblasts (Fitzgerald, 1979; Rutherford et al., 1993; Nakashima, 1994). Moreover, recent work has identified a population of human dental pulp stem cells capable of developing into odontoblasts (Gronthos et al., 2000; Shi et al., 2001).

An understanding of the characteristics of the cells that comprise the pulp, in particular the stromal cell, is taking on added importance in the context of emerging strategies for dental tissue engineering. One concept is that cells derived from the pulp may be expanded in culture for subsequent seeding into porous three-dimensional scaffolds to produce selected dental tissues in vitro or to be used in implants to facilitate regeneration in vivo (Mooney et al., 1996; Spector, 1998; Buurma et al., 1999). One phenotypic trait of stromal cells in other tissues that has not yet been investigated in pulp-derived cells in vitro is the expression of a muscle actin isoform, {alpha}-smooth-muscle actin (SMA).

Expression of SMA has been reported in bone marrow stromal cells comprising the adherent stem cell population in cultures of human (Charbord et al., 1990) and murine (Peled et al., 1991) bone marrow, and other work has demonstrated the ability of these cells to contract a collagen-glycosaminoglycan analog of extracellular matrix in vitro (Cai et al., 2001). These findings may explain the expression of SMA in, and the contraction of, musculoskeletal connective tissue cells presumably derived from marrow stromal stem cells (Spector, 2001). A recent investigation demonstrating that a stem cell population in human dental pulp has many of the phenotypic traits of these marrow-derived stem cells (Shi et al., 2001) prompts the study of SMA in pulp cells.

The objective of this study was to evaluate the expression of SMA in porcine dental pulp cells grown in monolayer culture and their contraction of a collagen-glycosaminoglycan sponge-like matrix commended by its value as a tissue regeneration template (Yannas et al., 1989) and as a useful analog of extracellular matrix for a contraction assay in vitro (Spector, 2001).


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Fabrication and Cross-linking of Collagen-Glycosaminoglycan Matrices
Porous three-dimensional collagen-glycosaminoglycan matrices were produced according to a previously described protocol (Yannas et al., 1989). In brief, type I collagen (bovine tendon, Integra LifeSciences, Plainsboro, NJ, USA) was blended with chondroitin 6-sulfate (Sigma Chem. Co., St. Louis, MO, USA). The slurry was frozen at -45°C and freeze-dried. The sponge-like scaffolds were subsequently cross-linked by dehydrothermal treatment for 24 hrs at 105°C and 30 mtorr (Yannas et al., 1989). The matrices have been previously reported to have a porosity of approximately 87% and an average pore size of 84 µm (Nehrer et al., 1997).

Dental Pulp Cell Harvest, Culture, and Passaging
The lower mandible from one pig, approximately 6 mos old, was obtained from an abattoir (Adams Farm, Athol, MA, USA). (Institutional Review Board approval was not needed for this study.) The mandible was soaked in butadiene for 15 min to prevent contamination and then rinsed in phosphate-buffered saline (PBS) before being transferred to a sterile environment. The bone overlying the unerupted premolars and molars was carefully chiseled away. An exposed third molar was gently removed from the bone and placed in a wash solution of PBS containing 1% antibiotics (100 µg/mL penicillin, 100 µg/mL streptomycin, and 2.5 µg/mL fungizone). The surrounding tissue was gently wiped away with sterile gauze. Enamel, dentin, and the odontoblastic layer were removed.

The dental pulp was removed from the sample, dissected into 2-mm3 specimens, and cultured in six-well plates. Approximately 0.5 mL of complete medium was added to the explants during the period allowed for tissue attachment. These cells were grown in Dulbecco's Eagle Medium/Nutrient Mixture F12 (DMEM/F12) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1% antibiotics (100 µg/mL penicillin, 100 µg/mL streptomycin, and 2.5 µg/mL fungizone), and 25 µg/mL ascorbic acid. Media were changed every other day, and the cells were cultured to confluence. At confluence, the cells were trypsinized and re-plated at a density of about 500,000 cells/75 cm2 for further growth for up to 3 passages. Aliquots of cells were stored in vials at –70°C.

Cell-seeded Collagen-Glycosaminoglycan Matrices
Matrix disks, 9 mm in diameter by 3.5 mm thick, were soaked in PBS for 1 hr at room temperature, and transferred to culture medium. Third-passage cells were counted by means of a hemacytometer and the concentration adjusted to 2 x 107/mL. Under sterile conditions, pre-wetted collagen-glycosaminoglycan matrices were lightly dried on sterile filter paper and placed on a six-well plate coated with 2% agarose. A 25-µL quantity of cell suspension was delivered to the surface of the collagen-glycosaminoglycan matrices. The matrices were flipped over, and another 25 µL of cell suspension was added to the opposite surface. The total number of cells per matrix was 1 x 106. Culture medium (0.5 mL) was added to each well, and the six-well plates were placed in the incubator at 37°C, 5% CO2, and 100% relative humidity for 3 hrs. Then, 2 mL of medium were added for a total of 2.5 mL per well. Medium was changed every other day.

We determined the diameter of the matrices by placing the culture dishes on a set of graduated circular templates. Measurements were made at 0 (n = 24), 1 (n = 24), 7 (n = 18), 14 (n = 12), and 28 (n = 6) days. Six samples were terminated after 1, 7, 14, and 28 days. Data were calculated as the percentage of the original diameter and as the percentage of cell-mediated contraction by subtraction of the percentage reduction of the diameter of the non-seeded matrices (n = 6) from the percentage decrease of the diameters of the cell-seeded specimens. This latter parameter was also normalized to the cell number reflected by the DNA content of the specimens measured at each of the 4 times of death (n = 4) with the use of the Hoechst 33258 dye (Polyscience Inc., Northampton, UK) method as previously reported (Cai et al., 2001).

Histology and Immunohistochemistry
Cell-seeded matrices recovered at each of the four times of death (n = 2) were fixed in formalin for 24 hrs, dehydrated in graded ethanol solutions, and embedded in paraffin. Sections, 6-7 µm in thickness, were cut with a microtome and stained with hematoxylin and eosin or allocated for SMA immunohistochemistry according to a previously reported method (Cai et al., 2001). The monoclonal anti-SMA antibody (Mouse Monoclonal anti-{alpha}-Smooth Muscle Actin, 1A4 Clone, Sigma #A2547, Sigma Chem. Co., St. Louis, MO, USA) used in this study binds to the amino terminal decapeptide of SMA (Skalli et al., 1986) and has been used in our own previously published studies (Spector, 2001) and in numerous other investigations for the immunolocalization of SMA in a wide variety of cell types (Charbord et al., 1990; Gronthos et al., 2000).

For the immunostaining of cells in monolayer, a modification of the method described above (Comut et al., 2000) was used.

Western Blot Analysis for SMA
Cells were trypsinized and protein extracted by a previously reported method (Comut et al., 2000). Samples containing 10 or 15 µg of total protein, a positive control of 5 µg total protein extracted from human aortic smooth-muscle cells, and a protein size marker were run. It was necessary to reduce the amounts of protein used from the higher-passage cells, because the SMA levels were so high that they blackened the film. The Western blot analysis with the SMA antibody described above followed a method previously reported (Comut et al., 2000).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Western Blot Analysis
SMA was detected in pulp cells immediately after their isolation (Fig. 1aGo). Of note was the increased SMA content of the cells with time in culture (i.e., with passage number; Fig. 1aGo). By the third passage, the pulp cells contained notably more SMA than the smooth-muscle-cell positive control (Fig. 1aGo).



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  		Figure 1. Western blot films and immunohistochemical micrographs demonstrating the presence of {alpha}-smooth-muscle actin (SMA) in pulp cells. (a) Western blot films showing the SMA content of freshly isolated pulp cells and cells after passages 1 and 3. The control band from the smooth-muscle cells controls is also shown for each run. The amount of protein used for each run is noted under the band. Less protein was used for the higher passage because the SMA content was so high that it blackened the film. (b-d) Immunohistochemistry of pulp cells in monolayer culture after the first (b) and third (d) passages showing the presence of SMA (darkly stained stress fibers) in many of the cells (b, d). The SMA can be seen incorporated into the prominent stress fibers (d). The negative control sample, absent the primary antibody (c), shows no sign of the chromogen. Scale bar, 50 µm.

 
The relatively large size of the cells (tens of micrometers in diameter) was in evidence, even during the first passage (Fig. 1bGo). With increasing time in culture, the cell size generally increased. The SMA content of the cells was also demonstrated by immunohistochemistry (Figs. 1b, 1dGo). Most cells in monolayer were seen to contain SMA after the first passage (Fig. 1bGo). By the third passage, the SMA could be seen to be incorporated into prominent stress fibers (Fig. 1dGo).

Histology and SMA Immunohistochemistry
Histology revealed that cells were dispersed throughout the matrix. As time in culture progressed, there was a dramatic decrease in the overall size of the cell-seeded specimens (documented in the next section) and a concomitant decrease in the pore diameter, as seen histologically (Fig. 2Go). By 7 days, there was an evident decrease, histologically, in the peripheral pore diameter, while the internal pores appeared to remain at their initial size (Fig. 2bGo). This differential pore compression was more obvious at 14 days (Fig. 2cGo). By 28 days in vitro, few open pores remained in the specimens (Figs. 2d, 3aGoGo). With increasing time in culture, the cells appeared to become entrapped in the contracting pores (Fig. 3aGo). Of importance was the fact that the histology was consistent with contracture, rather than dissolution being the principal cause of the dimensional change of the specimens (Schulz Torres et al., 2000).



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  		Figure 2. Histology of the pulp cell-seeded matrices after 1, 7, 14, and 28 days of culture, showing the contracture of the constructs with time in culture. Hematoxylin and eosin stain.

 


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  		Figure 3. Micrographs of the 28-day cell-seeded matrices stained with hematoxylin and eosin (a) and with the monoclonal antibody to SMA (b-d). The negative control for the immunohistochemistry is shown in the inset of (b).

 
Immunohistochemical staining of the tissue sections displayed the presence of SMA in cells at all of the time points of evaluation. The percentage of cells that contained SMA varied (Figs. 3b, 3c, 3dGo). In several regions, the majority of cells expressed this muscle actin isoform (Figs. 3b, 3c, 3dGo). There was no preference of SMA-positive or -negative cells for certain regions within the matrices. In selected cells, SMA was found co-localized with stress fibers (Fig. 3cGo).

Measured Change in the Collagen-Glycosaminoglycan Matrix Diameter with Time in Culture
There was a slight decrease in the outside diameter of the non-seeded collagen-glycosaminoglycan matrices with time in vitro (Fig. 4aGo), consistent with the mild plasticizing effect that medium has on the matrices (Schulz Torres et al., 2000). In contrast, the pulp cell-seeded specimens decreased in size to less than half of their original diameter by 28 days (Fig. 4aGo). A noticeable reduction in size was observed within the first day in culture (Fig. 4aGo). There appeared to be a relatively constant rate of contracture from 1 to 28 days (Fig. 4aGo). Two-factor analysis of variance (ANOVA) demonstrated that time and whether the matrices were seeded had significant effects on the diameter of the specimens (p < 0.0001 for each). Post hoc testing with Fisher's protected least-squares differences (PLSD) method showed that differences between the cell-seed and non-seeded groups and between groups at any two time points were statistically significant.



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  		Figure 4. Data demonstrating the pulp cell-mediated contraction of the collagen-glycosaminoglycan matrices. (a) Graph showing the reduction in diameter of the non-seeded and cell-seeded matrices with time in culture. Mean ± SEM. For the cell-seeded samples: 0 (n = 24), 1 (n = 24), 7 (n = 18), 14 (n = 12), and 28 (n = 6) days. For the non-seeded samples, n = 6. The error bars are smaller than the symbols used for the means. (b) Graph showing the DNA content of the matrices with time in culture (Mean ± SEM; n = 4). (c) Bar chart showing the cell-mediated contraction normalized to DNA content of the matrices. Mean ± SEM. 1 (n = 24), 7 (n = 18), 14 (n = 12), and 28 (n = 6) days.

 
The cell-mediated contraction, obtained by subtraction of the mean value of the contraction of the non-seeded matrices from the seeded matrices (Fig. 4aGo), increased from 20% of the original diameter at 1 day to 30% at 7 days, 40% at 14 days, and to more than 50% after 28 days in culture. Based on the histology of the 28-day specimens (Fig. 2dGo), little additional decrease in size might have been expected after 28 days.

The DNA content of the matrices doubled during the 28-day course of the experiment (Fig. 4bGo). Of interest was that the increase in DNA content that occurred from 1 to 14 days did so during the period when the matrices were undergoing the greatest amount of contracture. One-factor ANOVA revealed a significant effect of time in vitro on DNA content (p < 0.0001). Fisher's PLSD post hoc testing showed that differences between any two groups except the one- and seven-day time periods were statistically significant.

Cell-mediated contraction was normalized to the DNA content of the matrices, as a surrogate for cell number (Fig. 4cGo). This parameter increased from 1 to 7 days and slightly decreased thereafter, remaining constant through 28 days (Fig. 4cGo). One-factor ANOVA demonstrated a significant effect of time in culture on the cell-mediated contraction normalized to DNA content (p < 0.0001). Fisher's PLSD post hoc testing showed that differences between any two groups except the 14- and 28-day time periods were statistically significant.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This is the first investigation of the expression of SMA by cells derived from the dental pulp in vitro. SMA was detectable by Western blot analysis in pulp cells immediately after isolation. The levels of SMA increased with passage number. Two prior immunohistochemical investigations of papilla and pulp found SMA only in cells in the vessel walls (Lombardi et al., 1992; Pannone et al., 1998). However, the low number and narrow profile of stromal cells challenge their identification in immunohistochemical preparations in vivo. Another recent immunohistochemical study reported the presence of SMA in a population of human pulp cells in monolayer culture (Gronthos et al., 2000).

A notable finding of the present study was that the SMA-containing pulp cells had the capability to contract a collagen-glycosaminoglycan analog of extracellular matrix in vitro. The contractility of pulp cells can be placed in the context of the contractility of other cell types in collagen-glycosaminoglycan matrices of comparable composition, pore diameter, and size after similar times in culture. A recent study (Lee et al., 2001) of the contractility of SMA-containing third-passage adult canine chondrocytes reported 2%/µg DNA contraction of the matrix after 28 days. Also for comparison, using the same collagen-glycosaminoglycan scaffold, previous investigators (Schulz Torres et al., 2000) measured a contraction of 140%/µg DNA by calf tendon cells after 21 days. These comparisons suggest differences in the degree to which various SMA-expressing connective tissue cells can contract a collagen-glycosaminoglycan analog of extracellular matrix. These differences may be due to variations in the SMA content of the cells, the percentage of cells expressing SMA, their adhesion to the matrix, and the changes in the modulus of the matrix effected by the cells. How these findings relate to the behavior of these cells in vivo and in tissue engineering strategies are topics of ongoing work.

Prior work with other cells types has correlated the amount of SMA in the connective tissue cell with the degree of contraction (Hinz et al., 2001; Kinner and Spector, 2001). In one study, selected growth factors that up- and down-regulated SMA expression in chondrocytes were found to have a similar effect on the cell-mediated contraction of collagen-glycosaminoglycan scaffolds and the directly measured force of contraction (Zaleskas et al., 2001). Additional studies will be needed to investigate the effects of these and other agents on the SMA expression and contraction of pulp cells.

The culture method used in this study favored an adherent cell population in much the same way as the techniques used for the culture of stromal cells from tissues such as bone marrow (Friedenstein et al., 1976; Dexter et al., 1977; Haynesworth et al., 1992). One of the hallmarks of the marrow-derived pluripotent stem cell population is its large size and well-spread morphology, and its ability to proliferate in culture (Pittenger et al., 1999). The large size and prominent SMA-labeled stress fibers of the pulp cells replicated features of the stromal cells grown from marrow (Charbord et al., 1990; Peled et al., 1991). Of importance are recent studies reporting the similarity of pulp-derived stem cells with marrow stromal stem cells (Gronthos et al., 2000; Shi et al., 2001); the two cell types demonstrated a similar level of gene expression for more than 4000 known human genes (Shi et al., 2001). Future work needs to identify markers for better definition of pulp cells and confirm their kinship to stromal cells in other tissues, as well as their pluripotential capabilities.

Results of the present work suggest that SMA-enabled cell contraction can contribute to the contracture of pulp cell-seeded scaffolds like that reported for synthetic polymers (Mooney et al., 1996), as well as for the collagen scaffolds used in this study. It is clear that an unfavorable feature of this contraction is the distortion of the matrix (changes in shape and pore characteristics). This could compromise the use of the cell-seeded construct for tissue engineering. Future work needs to determine the factors affecting SMA expression in these cells and methods for its regulation. At the same time, this understanding could shed light on the role of SMA-dependent contraction of pulp cells in vivo and the impact of this phenotypic trait on their progeny.


   ACKNOWLEDGMENTS
 
This work was supported by the Harvard Craniofacial Tissue Engineering Center. The authors are grateful to Professor I.V. Yannas for use of the facilities in his Fibers and Polymers Laboratory at the Massachusetts Institute of Technology for the preparation of the collagen-glycosaminoglycan matrices. The histological assistance of Sandra Zaptka Taylor is greatly appreciated.

Received March 12, 2001; Last revision January 2, 2002; Accepted January 16, 2002


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Berkovitz BKB, Holland GR, Moxham BJ (1992). Color atlas and textbook of oral anatomy histology and embryology. London: Mosby-Year Book, Inc.

Buurma B, Gu K, Rutherford R (1999). Transplantation of human pulpal and gingival fibroblasts attached to synthetic scaffolds. Eur J Oral Sci 107:282–289.[Medline]

Cai D, Marty-Roix R, Hsu H-P, Spector M (2001). Lapine and canine bone marrow stromal cells contain smooth muscle actin and contract a collagen-glycosaminoglycan matrix. Tissue Eng 7:829–841.[Medline]

Charbord P, Lerat H, Newton I, Tamayo E, Gown AM, Singer JW, et al. (1990). The cytoskeleton of stromal cells from human bone marrow cultures resembles that of cultured smooth muscle cells. Exp Hematol 18:276–282.[Medline]

Comut AA, Shortkroff S, Zhang X, Spector M (2000). Association of fibroblast orientation around titanium in vitro with expression of a muscle actin. Biomaterials 21:1887–1896.[Medline]

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