0099-2399/92/1807-0327/$03.00/0 JOURNAL OF ENDODONTICS Copyright © 1992 by The American Association of Endodontists
Printed in U.S.A.
VOL. 18, NO. 7, JULY 1992
In Vivo and in Vitro Glycosaminoglycans from Human Dental Pulp Chutima Mangkornkarn, DDS, and James C. Steiner, DDS, MSD
main functions in the tooth is to regulate water retention in the pulp (2) and to protect the pulp tissues against high pressure. Other functions of GAG include formation and organization of collagen (3, 4), control of infection, inflammation, wound healing, cell proliferation, calcification, and as ion exchangers (5). Even though the GAG in dental pulps have been studied extensively in animals, a difference in the GAG content from various species has been clearly demonstrated (6-8). Furthermore, the types and distribution of GAG within the same species vary in different tissues in order to adapt to their specific biological functions (9). Knowledge of GAG in the human dental pulp is still limited and their actual role in the dental pulp is not fully understood. Controversy remains on the type of GAG found in human dental pulp. Chondroitin sulfate, hyaluronic acid, and dermatan sulfate have been consistently seen in human dental pulp tissue (7, 8, 10). Yet, Linde (7) and Embery (10) observed the presence of keratan sulfate while Sakamoto et al. (8) detected heparan sulfate (HS). The reasons for these discrepancies are not known. However, the method of GAG separation was thought to be a factor. Previous GAG analyses were done on large amounts of dental pulp tissues pooled from multiple human subjects (7, 8, 10). Utilizing a current technique, we are now able to extract and identify GAG from small amounts of freshly extracted pulp tissue from one individual. This would increase the accuracy of the analytical data. Existing knowledge regarding the GAG profile of human dental pulp has been derived primarily from biochemical studies (7, 8, 10). Since observations made from prepared tissues are static, they do not reveal the dynamic changes in living cells and tissues. Therefore, we decided to study the GAG synthesis by fibroblasts from the human dental pulp tissue. The in vitro model would allow us to study behavioral changes of the cells following injury or trauma. GAG synthesis from different fibroblastic passages were also compared. The purposes of this study were to (a) qualitatively determine the type of GAG present in normal human dental pulp; (b) determine and compare the GAG derived directly from dental pulp (in vivo) with those synthesized by cultured pulp fibroblasts from the same tissue (in vitro); and (c) determine and compare GAG synthesis between second and fourth passaged cultured pulp fibroblasts.
A qualitative assessment was made of the type of glycosaminoglycans (GAG) present in normal human dental pulp using eletrophoresis on celluloseacetate plates. A comparison was also made between the GAG derived directly from the dental pulp (in vivo) and those derived from cultured pulp fibroblasts from the same individual (in vitro). The results of this study showed four main types of GAG in normal human dental pulp tissue, which were dermatan sulfate, heparan sulfate, hyaluronic acid, and chondroitin sulfate. GAG synthesis from cultured pulp fibroblasts in vitro was different from the GAG present in the dental pulp (in vivo). Extracellular GAG, as well as pericellular GAG consisted of dermatan sulfate, hyaluronic acid, chondroitin sulfate, and heparin. Cellular GAG, however, contained only dermatan sulfate, hyaluronic acid, and chondroitin sulfate. There was no difference in type of GAG from the second and fourth passaged pulp fibroblasts.
The dental pulp is a highly specialized connective tissue which contains different types of cells, fibers, and amorphous ground substance (extracellular matrix). Residing in a rigid case, its survival mechanism through various types of trauma is most intriguing. The glycosaminoglycans (GAG), constituents in the extracellular matrix, are believed to play an important role in maintaining and regulating the function of all connective tissues as well as in the dental pulp. Since all of the nutrients and metabolites that go into or come out of the cell have to pass through the ground substance, any changes in the composition and physicochemical properties of GAG affect the physiological and pathological processes of the tissue (1). Structurally, GAG are high molecular weight mucopolysaccharides which are made up of repeating disaccharide units, each consisting of a hexosamine (glucosamine or galactosamine) and a hexuronic acid (Dglucuronic acid or L-iduronic acid), linked by glycosidic bands (Fig. 1). Naturally, GAG are present as a protein-carbohydrate complex or proteoglycan in tissues with many sulfated GAG side chains attached to the protein core. In biological fluids, GAG are present freely as individual units. This macromolecule is highly hydrophilic and polyanionic; thus, one of its
327
328
Journal of Endodontics
Mangkornkarn and Steiner coo-
o
cHaoso~
coo-
cHacO;
o ?4,o
=0
I
OH
I
.NSO;
I
o.
.NCOC.;
Heparan Sulphate (Tetrasaccharide) COOH
Ct"IzON
OH H
H
I
I
OH
HNCOCH3
H y a l u r o n i c Acid FIG 1. Demonstration of a disaccharide unit linked by a glycosidic bond. Repeating disaccharide units makes up the GAG unit. Various GAG show heterogeneity with respect to replacement of the glycosyl residues by acetyl or sulfate groups on the polymer and differences in linkage regions.
MATERIALS AND M E T H O D S Pulp Samples Ten patients between the age of 16 and 40 yr participated in this study. These patients elected to have their third molars removed in the Section of Oral Surgery, UCLA School of Dentistry. Pulp tissues were obtained from freshly extracted noncarious third molars. Each sample contained pulps from teeth obtained from the same patient. Any teeth which were cut during removal were excluded. Immediately after extraction the teeth were placed in 2x complete medium. The complete medium (2×) was a mixture of Dulbecco's modified Eagle's medium (1 x) containing 4500 mg/1 of D-glucose and L-glutamine (catalog no. 320-1965 AJ; GIBCO Laboratories, Grand Island, NY) supplemented with 10,000 units/ml penicillin base-10,000 ug/ml streptomycin, sodium pyruvate solution (1:50 vol/vol) (catalog no. 320-1360; GIBCO Laboratories), and 10% Nu-Serum (1:5 vol/vol) (catalog no. 5000; Collaborative Research, Bedford, MA). Attached soft tissues were removed from the root surfaces and the teeth were rinsed with the 2x complete medium to remove blood and tissue debris. The tooth was then cracked open with a sterile ViseGrip locking jaw plier. Pulp tissue was gently removed with an explorer and cotton pliers and then placed in a Petri dish with l x complete medium (1 x Dulbecco's modified Eagle's medium supplemented with 5000 units/ml penicillin base5000 #g/ml streptomycin base (catalog no. 11N02909; GIBCO Laboratories), sodium pyruvate solution (l: 100 vol/ vol), and 10% Nu-serum (l:10 vol/vol).
Tissue Preparation Pulp tissues from the molars of the same patient were diced into small pieces (1 to 2 mm 3) using sterile scissors and a scalpel. Tissue pieces were then divided into two parts. One half was used for isolation and analysis of GAG. The other
half was used for the culturing of pulp fibroblasts. The pulp tissue that was used for GAG analysis (pulpal GAG) was washed twice with 15 ml of Hanks' balanced salt solution (HBSS) (GIBCO Laboratories) and transferred into a 15-ml tube. The tissue was frozen until needed.
Cell Culture The other portion of pulp tissue was prepared immediately for culturing. Fibroblast cultures were established according to the method of Diegelmann et al. (11). Fresh human pulp tissue pieces were washed twice with 15 ml of 2x complete medium. They were then transferred into 5 ml of 1× complete medium, equally divided, and placed into two sterile 25-cm 2 tissue culture flasks. The flasks were incubated at 37°C for 2 to 3 wk to allow the cells to adhere to the flasks. Routine examination by light microscopy was done to ensure cell growth and confluency. When fibroblast cells became confluent, they were subcultivated by treatment with 0.05% trypsin-EDTA for 15 min at 37°C (trypsinization), centrifuged, and seeded into 75-cm z tissue culture flasks. The first passage culture was then established. The medium was changed when nutrients were exploited as indicated by a color change in the medium or after 7 days. The same procedure was used in passing the fibroblast cultures to the second, third, and fourth passages. Fibroblast cultures from the second and fourth passages were used for GAG analysis. Each culture to be analyzed was divided into three fractions. 1. Extracellular GAG were isolated from the culture medium. The medium was decanted and the cell layer washed with 5 ml of HBSS. The medium and one rinse were pooled and stored for GAG analysis. 2. Pericellular GAG were released from the cell layer by trypsinization with 0.05% trypsin-EDTA at 37"C for 15 min. Detached cells and trypsin-released material were collected and the plates washed with an additional 1 ml of HBSS. The rinse was added to the cell suspension. Following centrifuga-
Vol. 18, No. 7, July 1992
Glycosaminoglycans from Human Dental Pulp
tion at 2000 x g for 30 min, the supernatant was saved and the cell pellet washed with another 1 ml of HBSS. The combined supernatants comprised the pericelluIar fraction. 3. Cellular GAG were the remaining cells after trypsinization. The three fractions (extracellular, pericellular, and cellular GAG) were then digested with the pronase buffer (Behring Diagnostics, La Jolla, CA) (1 mg/50 ml of sample) and further analyzed as described below. Isolation of GAG
Frozen pulp tissue was allowed to dry under reduced pressure (lyophilization) for 24 to 48 h. Dried tissue pieces were digested with pronase buffer (1 mg/ml concentration) for 48 h at 37°C (12). Deactivation of pronase was done by boiling for 8 to 10 rain, and the solution was centrifuged at 2000 x g for 30 min to remove insoluble material. After digestion, GAG from the pulp tissue (pulpal GAG) and the three fractions from cultured fibroblasts were isolated from the suspensions by precipitation with cetylpyridinium chloride (13). The supernatants were dialyzed against water for 48 h, lyophilized, resuspended in 1% cetylpyridinium chloride in 0.02 M NaC1, and kept at room temperature for 48 h. After centrifugation at 2000 x g for 30 min, the pellets were resuspended in 1 ml of 2.0 M NaCI dissolved in 3 ml of absolute cold ethyl alcohol (100:15 vol/vol) and precipitated 48 h at 4"C. This step was repeated and the solutions were washed again and resuspended in 1 ml of distilled water and 3 ml of absolute cold ethyl alcohol for an additional 48 h at 4*C. After centrifugation, supernatants were discarded, cell pellets lyophilized, and resuspended in 15 ul of distilled water.
droitin sulfate (CS) (Fig. 2, pulp). The presence of HS was confirmed by the disappearance of HS band on the celluloseacetate plate when specific enzymatic digestion with heparitinase was used (Fig. 3). In vitro extracellular GAG, as well as pericellular GAG, contained heparin (HP), DS, HA, and CS (Fig. 2, E2, P2, E4, and P4). HS, which was present in pulpal GAG, was not present in any fractions derived from cultured fibroblasts. The cellular GAG, derived from digestion of cultured fibroblast cells, differed markedly from extracellular GAG, pericellular GAG, and pulpal GAG. Cellular GAG contained only DS, HA, and CS (Fig. 2, C2 and C4). Neither HS nor HP were detected in the cellular GAG fraction. Comparison of the GAG profile from the cultures of the second (E2, P2, and C2) and fourth (E4, P4, and C4) passages showed no difference in types and distribution of GAG in the
CS HA HS DS
:::~::::::&~
HP
Std
Analysis of GAG The samples were analyzed by electrophoresis on cellulose acetate plates (Helena Laboratories, Beaumont, TX) as described by Cappelletti et al. (14). GAG standards were coelectrophoresed. Phenol red (1 ~1) was added to each sample and standard as an indicator. Following electrophoresis, the membranes were stained with 0.1% Alcian blue and destained with multiple changes of 5.0% acetic acid. The types of GAG were identified by comparison of the bands present to those of reference standards. Selective digestion was also performed with heparitinase (ICN ImmunoBiotogicals, Lisle, IL) to confirm the presence of heparan sulfate (HS) which was controversial in previous studies (7, 8, 10). This enzyme specifically degrades heparan sulfate and leaving other GAG intact. The enzyme was also placed on GAG standard to ensure that the enzyme was functioning properly. Heparitinase digestion was performed using 0.001 units of heparitinase in 0.1 M calcium acetate (pH 7) at 40°C for 1 h. The samples were then boiled for 2 rain and compared by cellulose acetate electrophoresis to identify a specific GAG component. RESULTS Electrophoretic analysis of normal human dental pulp tissue indicated the presence of four types of GAG which were dermatan sulfate (DS), HS, hyaluronic acid (HA), and chon-
329
Pulp
E2
Pz
C2
E4
P4
C4
FIG 2. Diagramatic representation of cellulose acetate electrophoresis of GAG isolated from normal human dental pulp. Std, GAG standards; pulp, pulpal GAG; E2, extracellular GAG from second passage culture; P2, pericellular GAG from second passage culture; C2, cellular GAG from second passage culture; E,, extracellular GAG from the fourth passaged culture; P4, pericellular GAG from the fourth passage culture; C4, cellular GAG from fourth passage culture. The migration of GAG are shown.
CS HA
HP
' .~
Sld,
'.~:f
Pulp I
Pulp I
& Enzyme
Pulp II
Pulp lI
& Enzyme
FIG 3. Cellulose acetate electrophoresis of pulpal GAG with and without heparitinase digestion. Std, standard GAG; pulp, pulpal GAG; pulp + enzyme, pulpal GAG with enzyme digestion. HS band disappears from the samples with addition of heparitinase enz.
330
Mangkornkarn and Steiner
extracellular and pericellular fractions of cultured fibroblasts. Cellular GAG from the second and fourth passaged fibroblasts contained similar components. Both cellular GAG were different from the extracellular and pericellular GAG (Fig. 2). When the in vitro GAG from all fractions were combined and compared with those found in vivo. GAG from the cultured pulp fibroblasts were apparently different from those derived directly from the pulp tissue (in vivo). HP was consistently shown in vitro while HS was present in vivo (Fig. 2). DISCUSSION The results of this study demonstrate four main types of GAG in normal human dental pulps which are HS, DS, HA, and CS. This fnding supports the study of Sakamoto et al. (8) which detected the same types of GAG from human intact mandibular third molars. However, it is not consistent with other studies (7, 10) which reported only three types of GAG (DS, HA, and CS) in human dental pulp. Whether the type of tooth studied is responsible for the discrepancies is not clear. Both the Sakamoto et al. study (8) and our studies used intact mandibular molars. While Linde (7) used only premolars, Embery (10) used dental pulps from both premolars and molars and did not detect HS. Therefore, the tooth type does not appear to be the reason for the difference. Our study, as well as that of Sakamoto et al. (8), also seems to indicate that the isolation technique is not responsible for the difference in GAG identifcation. Both Linde (7) and Embery (10) used fixed pulp tissue pooled from multiple patients. It is possible that the fixation process, as opposed to the use of fresh tissues, may prevent complete GAG separation. The incompleteness of GAG separation was also suggested by Sakamoto et al. (8) to cause the variation in results. Although electrophoresis is used extensively to analyze GAG, interpretation of the data is sometimes equivocal. The proximity among specific bands (i.e. keratan sulfate, heparan sulfate) posed a problem in identifying the type of GAG (7). The pH of the electrophoretic buffer is now known to affect the band position. The buffer in this study was set to pH 5 to allow a clearer separation of all bands, especially between keratan sulfate and HS. In addition, selective enzyme digestion was used to reconfirm the type of GAG present on the cellulose-acetate plate. Heparitinase enzyme was used to confirm the presence of HS. Heparitinase selectively digests HS without disturbing any other GAG. The disappearance of the HS band from both standard and experimental columns proves that HS is present in normal human dental pulp tissue (Fig. 3). In vitro GAG synthesis by pulp fibroblasts was studied for the first time in this study. GAG types were separately identified in the extracellular, pericellular, and cellular fractions of cultured pulp fibroblasts, and different GAG components were found between the three fractions. Extracellular, as well as pericellular GAG, contained HP, DS, HA, and CS. Cellular GAG, however, contained only DS, HA, and CS. It is an interesting observation that HP synthesized by cultured pulp fibroblasts appear to be located only at the periphery. HP generally occurs intracellularly in the granules of mast cells and is released into the extracellular matrix of the tissue under the influence of certain stimuli. Since there were no mast cells in culture, the result of this study suggests that pulp fibroblasts may have the ability to synthesize HP.
Journal of Endodontics
When the GAG from the extracellular, pericellular, and cellular fractions of cultured pulp fibroblasts were combined together to represent total GAG produced in vitro, the primary difference observed was the presence of HP as opposed to HS in pulpal GAG (in vivo). It is possible that pulpal GAG are derived from a variety of cells and tissues in the dental pulp, i.e., fibroblasts, odontoblasts, mast cells, blood cells, and so forth, whereas the GAG isolated in vitro are from pure cultured fibroblasts. However, the lack of HP synthesis in vivo indicates that pulp fibroblasts do not synthesize HP in the normal state; neither do mast cells, although they have been reported to be residents of normal human dental pulp (15). The reason for these differences is not known at the present time. Multienzyme systems found in different locations of the cell or other unknown factors that regulate the relative activities of the various enzymes are believed to control the biosynthesis of heparin or heparan sulfate (16). It is speculated that the enzymes, as well as minerals and vitamins, mainly contribute to the regulation of GAG biosynthesis. The results of this study suggest that the use of in vitro pulp fibroblasts as a model to study biochemical changes of the dental pulp may not be as useful as originally assumed. Any interpretation of in vitro experimental results must be done with caution as it may be misleading. The demonstrated differences, however, may prove more important in developing additional methods for studying the biochemical changes of dental pulp in vivo. Although HP synthesis is caused by stimulation of mast cells, heparan sulfate is believed to be a ubiquitous component of cell surfaces in most or all cell types (17). Both HS and HP contain equimolar portions of glucosamine and hexuronic acid. However, HS is characterized by a higher content of Nacetyl groups than HP and glucuronic acid rather than iduronic acid. Despite the similarity of their polysaccharide component, HP and HS are genetically distinct with respect to their core proteins (16). In addition, HS has a smaller amount of sulfate groups than HP. These factors affect the physicochemical properties of both macromolecules. Murata (18) suggested that the HS macromolecule was involved in the transport of cations. Hjerpe and Engfeldt (19) suggested that HS, as a strong anionic molecule, may participate in the mineralization process. The presence of HS in the pulp in this study and its presence in human predentin and dentin (20) suggests the possible role of HS in the formation and repair of dentin. Since cultured pulp fibroblasts do not synthesize HS, it is possible that the odontoblasts may play an important role in I-IS synthesis. Finally, this study did not find any difference in the type of GAG between different passages; the extracellular and pericellular GAG from the second passaged fibroblasts showed similar GAG components to the extracellular and pericellular GAG from the fourth passaged fibroblasts. There was also no difference in the cellular GAG from both passages. This indicated that the cultures' environmental conditions do not influence the behaviour of in vitro cultured fibroblasts from the second to the fourth passages. This investigation was supported by the Dental Research Institute, UCLA School of Dentistry. We would like to express our sincere appreciation to Duncan Ellis, Dr. Diana Messadi, and Steven Berg for their technical assistance. Thanks to Dr. Gary Massa for his review of the manuscript. We also thank Dr. Charles Bertolami
Glycosaminoglycans from Human Dental Pulp
Vol. 18, No. 7, July 1992 and the UCLA Oral Surgery Section for provision of teeth and the laboratory space. Dr. Mangkornkarn is an adjunct assistant professor, Section of Endodontics, UCLA School of Dentistry, Los Angeles, CA. Dr. Steiner is chairman and assistant professor, Section of Endodontics, UCLA School of Dentistry. Address requests for reprints to Dr. Chutima Mangkornkarn, Section of Endodontics, 23-087 CHS, UCLA School of Dentistry, 10833 Le Conte Avenue, Los Angeles, CA 90024-1668.
References 1. Fricke R, Hartman F. Connective tissues. Biochemistry and pathophysiology. New York: Springer Verlag, 1974. 2. Fessler JH. A structural function of mucopolysaccharides in connective tissue. Biochem J 1960;76:124-32. 3. Bachra BN. Calcification, A problem in molecular biology. Adv Fluorine Res Dent Caries Prev 1966;4:95-101. 4. Lennox DW, Provenza DV. Mucopolysaccharides in odontogenesis. Histochemistry 1970;23:328-41. 5. Varma R, Varma RS. Mucopolysaccharides of body fluids in health and disease. New York: Walter de Gruyter, 1983:91-110. 6. Linde A. Glycosaminoglycans (mucopolysacchaddes) of the porcine dental pulp. Arch Oral Bio11970;15:1035-46. 7. Linde A. A study of the dental pulp glycosaminoglycans from permanent human teeth, and rat and rabbit incisor. Arch Oral Bio11973;18:49-9. 8. Sakamoto N, Okamoto H, Okuda K. Qualitative and quantitative analyses of bovine, rabbit and human dental pulp glycosaminoglycans. J Dent Res 1979;58:646-55.
331
9. Bronson RE, Argenta JG, Siebert EP, Bertolami CN. Distinctive fibrobiastic subpopulations in skin and oral mucosa demonstrated by differences in glycosaminoglycan content. In Vitro Cell Dev Biol 1988;24:1121-6. 10. Embery G. Glycosaminoglycans of human dental pulp. J Biol Buccale 1976;4:229-36. 11. Diegelmann RF, Cohen I, McCoy B. Growth kinetics and collagen synthesis of normal skin, normal scar and keloid fibroblasts in vitro. Cell Physiol 1979;98:341-6. 12. Savage KE, Swann DA. A comparison of glycosaminoglycan synthesis by human fibroblasts from normal skin, normal scar and hypertrophic scar. J Invest Dermatol 1985;84:521-6. 13. Scott JE. The preparation and fractionafion of acid polysaccharides using long-chain quarternary ammonium compounds. Biochem J 1956;62:31. 14. Cappelletti R, Del Rosso R, Chiarugi VP. A new electrophoretic method for the complete separation of all known glycosaminoglycens in a monodimensional run. Anal Biochem 1979;99:311-5. 15. Farnoush A. Mast cells in human dental pulp. J Endodon 1987;13:3623. 16. Roden L. Structure and metabolism of connective tissue proteoglycans. In: Lennarz W J, ed. The biochemistry of glycoproteins and proteoglycans. New York: Plenum Press, 1980:267-371. 17. Kraemer PM. Heparan sulphate of cultured cells. I. Membrane-associated and cell sap species in Chinese hamster cells. Biochemistry 1971;10:1437-45. 18. Murata K. Acidic glycosaminoglycans in human kidney tissue. Clin Chim Acta 1975;63:157-69. 19. Hjerpe A, Engfeldt B. Proteoglycans of dentine and predentine. Calcif Tissue Res 1976;22:173-82. 20. Branford White CJ. Molecular organization of heparan sulphate proteoglycan from human dentine. Arch Oral Bio11978;23:1141-4.
The Way It Was In Kingston, New Hampshire, in 1730 a hog owned by a Mr. Clough was "taken sick of a complaint in the throat and died." Mr. CIough butchered the hog and subsequently also died. By the fall of 1731, 802 children under the age of 10 were dead from airway obstruction caused by "extensive membranous inflammation of the throat." Epidemic diphtheria had arrived in New England. William Cornelius
Printed in U.S.A.
VOL. 18, NO. 7, JULY 1992
In Vivo and in Vitro Glycosaminoglycans from Human Dental Pulp Chutima Mangkornkarn, DDS, and James C. Steiner, DDS, MSD
main functions in the tooth is to regulate water retention in the pulp (2) and to protect the pulp tissues against high pressure. Other functions of GAG include formation and organization of collagen (3, 4), control of infection, inflammation, wound healing, cell proliferation, calcification, and as ion exchangers (5). Even though the GAG in dental pulps have been studied extensively in animals, a difference in the GAG content from various species has been clearly demonstrated (6-8). Furthermore, the types and distribution of GAG within the same species vary in different tissues in order to adapt to their specific biological functions (9). Knowledge of GAG in the human dental pulp is still limited and their actual role in the dental pulp is not fully understood. Controversy remains on the type of GAG found in human dental pulp. Chondroitin sulfate, hyaluronic acid, and dermatan sulfate have been consistently seen in human dental pulp tissue (7, 8, 10). Yet, Linde (7) and Embery (10) observed the presence of keratan sulfate while Sakamoto et al. (8) detected heparan sulfate (HS). The reasons for these discrepancies are not known. However, the method of GAG separation was thought to be a factor. Previous GAG analyses were done on large amounts of dental pulp tissues pooled from multiple human subjects (7, 8, 10). Utilizing a current technique, we are now able to extract and identify GAG from small amounts of freshly extracted pulp tissue from one individual. This would increase the accuracy of the analytical data. Existing knowledge regarding the GAG profile of human dental pulp has been derived primarily from biochemical studies (7, 8, 10). Since observations made from prepared tissues are static, they do not reveal the dynamic changes in living cells and tissues. Therefore, we decided to study the GAG synthesis by fibroblasts from the human dental pulp tissue. The in vitro model would allow us to study behavioral changes of the cells following injury or trauma. GAG synthesis from different fibroblastic passages were also compared. The purposes of this study were to (a) qualitatively determine the type of GAG present in normal human dental pulp; (b) determine and compare the GAG derived directly from dental pulp (in vivo) with those synthesized by cultured pulp fibroblasts from the same tissue (in vitro); and (c) determine and compare GAG synthesis between second and fourth passaged cultured pulp fibroblasts.
A qualitative assessment was made of the type of glycosaminoglycans (GAG) present in normal human dental pulp using eletrophoresis on celluloseacetate plates. A comparison was also made between the GAG derived directly from the dental pulp (in vivo) and those derived from cultured pulp fibroblasts from the same individual (in vitro). The results of this study showed four main types of GAG in normal human dental pulp tissue, which were dermatan sulfate, heparan sulfate, hyaluronic acid, and chondroitin sulfate. GAG synthesis from cultured pulp fibroblasts in vitro was different from the GAG present in the dental pulp (in vivo). Extracellular GAG, as well as pericellular GAG consisted of dermatan sulfate, hyaluronic acid, chondroitin sulfate, and heparin. Cellular GAG, however, contained only dermatan sulfate, hyaluronic acid, and chondroitin sulfate. There was no difference in type of GAG from the second and fourth passaged pulp fibroblasts.
The dental pulp is a highly specialized connective tissue which contains different types of cells, fibers, and amorphous ground substance (extracellular matrix). Residing in a rigid case, its survival mechanism through various types of trauma is most intriguing. The glycosaminoglycans (GAG), constituents in the extracellular matrix, are believed to play an important role in maintaining and regulating the function of all connective tissues as well as in the dental pulp. Since all of the nutrients and metabolites that go into or come out of the cell have to pass through the ground substance, any changes in the composition and physicochemical properties of GAG affect the physiological and pathological processes of the tissue (1). Structurally, GAG are high molecular weight mucopolysaccharides which are made up of repeating disaccharide units, each consisting of a hexosamine (glucosamine or galactosamine) and a hexuronic acid (Dglucuronic acid or L-iduronic acid), linked by glycosidic bands (Fig. 1). Naturally, GAG are present as a protein-carbohydrate complex or proteoglycan in tissues with many sulfated GAG side chains attached to the protein core. In biological fluids, GAG are present freely as individual units. This macromolecule is highly hydrophilic and polyanionic; thus, one of its
327
328
Journal of Endodontics
Mangkornkarn and Steiner coo-
o
cHaoso~
coo-
cHacO;
o ?4,o
=0
I
OH
I
.NSO;
I
o.
.NCOC.;
Heparan Sulphate (Tetrasaccharide) COOH
Ct"IzON
OH H
H
I
I
OH
HNCOCH3
H y a l u r o n i c Acid FIG 1. Demonstration of a disaccharide unit linked by a glycosidic bond. Repeating disaccharide units makes up the GAG unit. Various GAG show heterogeneity with respect to replacement of the glycosyl residues by acetyl or sulfate groups on the polymer and differences in linkage regions.
MATERIALS AND M E T H O D S Pulp Samples Ten patients between the age of 16 and 40 yr participated in this study. These patients elected to have their third molars removed in the Section of Oral Surgery, UCLA School of Dentistry. Pulp tissues were obtained from freshly extracted noncarious third molars. Each sample contained pulps from teeth obtained from the same patient. Any teeth which were cut during removal were excluded. Immediately after extraction the teeth were placed in 2x complete medium. The complete medium (2×) was a mixture of Dulbecco's modified Eagle's medium (1 x) containing 4500 mg/1 of D-glucose and L-glutamine (catalog no. 320-1965 AJ; GIBCO Laboratories, Grand Island, NY) supplemented with 10,000 units/ml penicillin base-10,000 ug/ml streptomycin, sodium pyruvate solution (1:50 vol/vol) (catalog no. 320-1360; GIBCO Laboratories), and 10% Nu-Serum (1:5 vol/vol) (catalog no. 5000; Collaborative Research, Bedford, MA). Attached soft tissues were removed from the root surfaces and the teeth were rinsed with the 2x complete medium to remove blood and tissue debris. The tooth was then cracked open with a sterile ViseGrip locking jaw plier. Pulp tissue was gently removed with an explorer and cotton pliers and then placed in a Petri dish with l x complete medium (1 x Dulbecco's modified Eagle's medium supplemented with 5000 units/ml penicillin base5000 #g/ml streptomycin base (catalog no. 11N02909; GIBCO Laboratories), sodium pyruvate solution (l: 100 vol/ vol), and 10% Nu-serum (l:10 vol/vol).
Tissue Preparation Pulp tissues from the molars of the same patient were diced into small pieces (1 to 2 mm 3) using sterile scissors and a scalpel. Tissue pieces were then divided into two parts. One half was used for isolation and analysis of GAG. The other
half was used for the culturing of pulp fibroblasts. The pulp tissue that was used for GAG analysis (pulpal GAG) was washed twice with 15 ml of Hanks' balanced salt solution (HBSS) (GIBCO Laboratories) and transferred into a 15-ml tube. The tissue was frozen until needed.
Cell Culture The other portion of pulp tissue was prepared immediately for culturing. Fibroblast cultures were established according to the method of Diegelmann et al. (11). Fresh human pulp tissue pieces were washed twice with 15 ml of 2x complete medium. They were then transferred into 5 ml of 1× complete medium, equally divided, and placed into two sterile 25-cm 2 tissue culture flasks. The flasks were incubated at 37°C for 2 to 3 wk to allow the cells to adhere to the flasks. Routine examination by light microscopy was done to ensure cell growth and confluency. When fibroblast cells became confluent, they were subcultivated by treatment with 0.05% trypsin-EDTA for 15 min at 37°C (trypsinization), centrifuged, and seeded into 75-cm z tissue culture flasks. The first passage culture was then established. The medium was changed when nutrients were exploited as indicated by a color change in the medium or after 7 days. The same procedure was used in passing the fibroblast cultures to the second, third, and fourth passages. Fibroblast cultures from the second and fourth passages were used for GAG analysis. Each culture to be analyzed was divided into three fractions. 1. Extracellular GAG were isolated from the culture medium. The medium was decanted and the cell layer washed with 5 ml of HBSS. The medium and one rinse were pooled and stored for GAG analysis. 2. Pericellular GAG were released from the cell layer by trypsinization with 0.05% trypsin-EDTA at 37"C for 15 min. Detached cells and trypsin-released material were collected and the plates washed with an additional 1 ml of HBSS. The rinse was added to the cell suspension. Following centrifuga-
Vol. 18, No. 7, July 1992
Glycosaminoglycans from Human Dental Pulp
tion at 2000 x g for 30 min, the supernatant was saved and the cell pellet washed with another 1 ml of HBSS. The combined supernatants comprised the pericelluIar fraction. 3. Cellular GAG were the remaining cells after trypsinization. The three fractions (extracellular, pericellular, and cellular GAG) were then digested with the pronase buffer (Behring Diagnostics, La Jolla, CA) (1 mg/50 ml of sample) and further analyzed as described below. Isolation of GAG
Frozen pulp tissue was allowed to dry under reduced pressure (lyophilization) for 24 to 48 h. Dried tissue pieces were digested with pronase buffer (1 mg/ml concentration) for 48 h at 37°C (12). Deactivation of pronase was done by boiling for 8 to 10 rain, and the solution was centrifuged at 2000 x g for 30 min to remove insoluble material. After digestion, GAG from the pulp tissue (pulpal GAG) and the three fractions from cultured fibroblasts were isolated from the suspensions by precipitation with cetylpyridinium chloride (13). The supernatants were dialyzed against water for 48 h, lyophilized, resuspended in 1% cetylpyridinium chloride in 0.02 M NaC1, and kept at room temperature for 48 h. After centrifugation at 2000 x g for 30 min, the pellets were resuspended in 1 ml of 2.0 M NaCI dissolved in 3 ml of absolute cold ethyl alcohol (100:15 vol/vol) and precipitated 48 h at 4"C. This step was repeated and the solutions were washed again and resuspended in 1 ml of distilled water and 3 ml of absolute cold ethyl alcohol for an additional 48 h at 4*C. After centrifugation, supernatants were discarded, cell pellets lyophilized, and resuspended in 15 ul of distilled water.
droitin sulfate (CS) (Fig. 2, pulp). The presence of HS was confirmed by the disappearance of HS band on the celluloseacetate plate when specific enzymatic digestion with heparitinase was used (Fig. 3). In vitro extracellular GAG, as well as pericellular GAG, contained heparin (HP), DS, HA, and CS (Fig. 2, E2, P2, E4, and P4). HS, which was present in pulpal GAG, was not present in any fractions derived from cultured fibroblasts. The cellular GAG, derived from digestion of cultured fibroblast cells, differed markedly from extracellular GAG, pericellular GAG, and pulpal GAG. Cellular GAG contained only DS, HA, and CS (Fig. 2, C2 and C4). Neither HS nor HP were detected in the cellular GAG fraction. Comparison of the GAG profile from the cultures of the second (E2, P2, and C2) and fourth (E4, P4, and C4) passages showed no difference in types and distribution of GAG in the
CS HA HS DS
:::~::::::&~
HP
Std
Analysis of GAG The samples were analyzed by electrophoresis on cellulose acetate plates (Helena Laboratories, Beaumont, TX) as described by Cappelletti et al. (14). GAG standards were coelectrophoresed. Phenol red (1 ~1) was added to each sample and standard as an indicator. Following electrophoresis, the membranes were stained with 0.1% Alcian blue and destained with multiple changes of 5.0% acetic acid. The types of GAG were identified by comparison of the bands present to those of reference standards. Selective digestion was also performed with heparitinase (ICN ImmunoBiotogicals, Lisle, IL) to confirm the presence of heparan sulfate (HS) which was controversial in previous studies (7, 8, 10). This enzyme specifically degrades heparan sulfate and leaving other GAG intact. The enzyme was also placed on GAG standard to ensure that the enzyme was functioning properly. Heparitinase digestion was performed using 0.001 units of heparitinase in 0.1 M calcium acetate (pH 7) at 40°C for 1 h. The samples were then boiled for 2 rain and compared by cellulose acetate electrophoresis to identify a specific GAG component. RESULTS Electrophoretic analysis of normal human dental pulp tissue indicated the presence of four types of GAG which were dermatan sulfate (DS), HS, hyaluronic acid (HA), and chon-
329
Pulp
E2
Pz
C2
E4
P4
C4
FIG 2. Diagramatic representation of cellulose acetate electrophoresis of GAG isolated from normal human dental pulp. Std, GAG standards; pulp, pulpal GAG; E2, extracellular GAG from second passage culture; P2, pericellular GAG from second passage culture; C2, cellular GAG from second passage culture; E,, extracellular GAG from the fourth passaged culture; P4, pericellular GAG from the fourth passage culture; C4, cellular GAG from fourth passage culture. The migration of GAG are shown.
CS HA
HP
' .~
Sld,
'.~:f
Pulp I
Pulp I
& Enzyme
Pulp II
Pulp lI
& Enzyme
FIG 3. Cellulose acetate electrophoresis of pulpal GAG with and without heparitinase digestion. Std, standard GAG; pulp, pulpal GAG; pulp + enzyme, pulpal GAG with enzyme digestion. HS band disappears from the samples with addition of heparitinase enz.
330
Mangkornkarn and Steiner
extracellular and pericellular fractions of cultured fibroblasts. Cellular GAG from the second and fourth passaged fibroblasts contained similar components. Both cellular GAG were different from the extracellular and pericellular GAG (Fig. 2). When the in vitro GAG from all fractions were combined and compared with those found in vivo. GAG from the cultured pulp fibroblasts were apparently different from those derived directly from the pulp tissue (in vivo). HP was consistently shown in vitro while HS was present in vivo (Fig. 2). DISCUSSION The results of this study demonstrate four main types of GAG in normal human dental pulps which are HS, DS, HA, and CS. This fnding supports the study of Sakamoto et al. (8) which detected the same types of GAG from human intact mandibular third molars. However, it is not consistent with other studies (7, 10) which reported only three types of GAG (DS, HA, and CS) in human dental pulp. Whether the type of tooth studied is responsible for the discrepancies is not clear. Both the Sakamoto et al. study (8) and our studies used intact mandibular molars. While Linde (7) used only premolars, Embery (10) used dental pulps from both premolars and molars and did not detect HS. Therefore, the tooth type does not appear to be the reason for the difference. Our study, as well as that of Sakamoto et al. (8), also seems to indicate that the isolation technique is not responsible for the difference in GAG identifcation. Both Linde (7) and Embery (10) used fixed pulp tissue pooled from multiple patients. It is possible that the fixation process, as opposed to the use of fresh tissues, may prevent complete GAG separation. The incompleteness of GAG separation was also suggested by Sakamoto et al. (8) to cause the variation in results. Although electrophoresis is used extensively to analyze GAG, interpretation of the data is sometimes equivocal. The proximity among specific bands (i.e. keratan sulfate, heparan sulfate) posed a problem in identifying the type of GAG (7). The pH of the electrophoretic buffer is now known to affect the band position. The buffer in this study was set to pH 5 to allow a clearer separation of all bands, especially between keratan sulfate and HS. In addition, selective enzyme digestion was used to reconfirm the type of GAG present on the cellulose-acetate plate. Heparitinase enzyme was used to confirm the presence of HS. Heparitinase selectively digests HS without disturbing any other GAG. The disappearance of the HS band from both standard and experimental columns proves that HS is present in normal human dental pulp tissue (Fig. 3). In vitro GAG synthesis by pulp fibroblasts was studied for the first time in this study. GAG types were separately identified in the extracellular, pericellular, and cellular fractions of cultured pulp fibroblasts, and different GAG components were found between the three fractions. Extracellular, as well as pericellular GAG, contained HP, DS, HA, and CS. Cellular GAG, however, contained only DS, HA, and CS. It is an interesting observation that HP synthesized by cultured pulp fibroblasts appear to be located only at the periphery. HP generally occurs intracellularly in the granules of mast cells and is released into the extracellular matrix of the tissue under the influence of certain stimuli. Since there were no mast cells in culture, the result of this study suggests that pulp fibroblasts may have the ability to synthesize HP.
Journal of Endodontics
When the GAG from the extracellular, pericellular, and cellular fractions of cultured pulp fibroblasts were combined together to represent total GAG produced in vitro, the primary difference observed was the presence of HP as opposed to HS in pulpal GAG (in vivo). It is possible that pulpal GAG are derived from a variety of cells and tissues in the dental pulp, i.e., fibroblasts, odontoblasts, mast cells, blood cells, and so forth, whereas the GAG isolated in vitro are from pure cultured fibroblasts. However, the lack of HP synthesis in vivo indicates that pulp fibroblasts do not synthesize HP in the normal state; neither do mast cells, although they have been reported to be residents of normal human dental pulp (15). The reason for these differences is not known at the present time. Multienzyme systems found in different locations of the cell or other unknown factors that regulate the relative activities of the various enzymes are believed to control the biosynthesis of heparin or heparan sulfate (16). It is speculated that the enzymes, as well as minerals and vitamins, mainly contribute to the regulation of GAG biosynthesis. The results of this study suggest that the use of in vitro pulp fibroblasts as a model to study biochemical changes of the dental pulp may not be as useful as originally assumed. Any interpretation of in vitro experimental results must be done with caution as it may be misleading. The demonstrated differences, however, may prove more important in developing additional methods for studying the biochemical changes of dental pulp in vivo. Although HP synthesis is caused by stimulation of mast cells, heparan sulfate is believed to be a ubiquitous component of cell surfaces in most or all cell types (17). Both HS and HP contain equimolar portions of glucosamine and hexuronic acid. However, HS is characterized by a higher content of Nacetyl groups than HP and glucuronic acid rather than iduronic acid. Despite the similarity of their polysaccharide component, HP and HS are genetically distinct with respect to their core proteins (16). In addition, HS has a smaller amount of sulfate groups than HP. These factors affect the physicochemical properties of both macromolecules. Murata (18) suggested that the HS macromolecule was involved in the transport of cations. Hjerpe and Engfeldt (19) suggested that HS, as a strong anionic molecule, may participate in the mineralization process. The presence of HS in the pulp in this study and its presence in human predentin and dentin (20) suggests the possible role of HS in the formation and repair of dentin. Since cultured pulp fibroblasts do not synthesize HS, it is possible that the odontoblasts may play an important role in I-IS synthesis. Finally, this study did not find any difference in the type of GAG between different passages; the extracellular and pericellular GAG from the second passaged fibroblasts showed similar GAG components to the extracellular and pericellular GAG from the fourth passaged fibroblasts. There was also no difference in the cellular GAG from both passages. This indicated that the cultures' environmental conditions do not influence the behaviour of in vitro cultured fibroblasts from the second to the fourth passages. This investigation was supported by the Dental Research Institute, UCLA School of Dentistry. We would like to express our sincere appreciation to Duncan Ellis, Dr. Diana Messadi, and Steven Berg for their technical assistance. Thanks to Dr. Gary Massa for his review of the manuscript. We also thank Dr. Charles Bertolami
Glycosaminoglycans from Human Dental Pulp
Vol. 18, No. 7, July 1992 and the UCLA Oral Surgery Section for provision of teeth and the laboratory space. Dr. Mangkornkarn is an adjunct assistant professor, Section of Endodontics, UCLA School of Dentistry, Los Angeles, CA. Dr. Steiner is chairman and assistant professor, Section of Endodontics, UCLA School of Dentistry. Address requests for reprints to Dr. Chutima Mangkornkarn, Section of Endodontics, 23-087 CHS, UCLA School of Dentistry, 10833 Le Conte Avenue, Los Angeles, CA 90024-1668.
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9. Bronson RE, Argenta JG, Siebert EP, Bertolami CN. Distinctive fibrobiastic subpopulations in skin and oral mucosa demonstrated by differences in glycosaminoglycan content. In Vitro Cell Dev Biol 1988;24:1121-6. 10. Embery G. Glycosaminoglycans of human dental pulp. J Biol Buccale 1976;4:229-36. 11. Diegelmann RF, Cohen I, McCoy B. Growth kinetics and collagen synthesis of normal skin, normal scar and keloid fibroblasts in vitro. Cell Physiol 1979;98:341-6. 12. Savage KE, Swann DA. A comparison of glycosaminoglycan synthesis by human fibroblasts from normal skin, normal scar and hypertrophic scar. J Invest Dermatol 1985;84:521-6. 13. Scott JE. The preparation and fractionafion of acid polysaccharides using long-chain quarternary ammonium compounds. Biochem J 1956;62:31. 14. Cappelletti R, Del Rosso R, Chiarugi VP. A new electrophoretic method for the complete separation of all known glycosaminoglycens in a monodimensional run. Anal Biochem 1979;99:311-5. 15. Farnoush A. Mast cells in human dental pulp. J Endodon 1987;13:3623. 16. Roden L. Structure and metabolism of connective tissue proteoglycans. In: Lennarz W J, ed. The biochemistry of glycoproteins and proteoglycans. New York: Plenum Press, 1980:267-371. 17. Kraemer PM. Heparan sulphate of cultured cells. I. Membrane-associated and cell sap species in Chinese hamster cells. Biochemistry 1971;10:1437-45. 18. Murata K. Acidic glycosaminoglycans in human kidney tissue. Clin Chim Acta 1975;63:157-69. 19. Hjerpe A, Engfeldt B. Proteoglycans of dentine and predentine. Calcif Tissue Res 1976;22:173-82. 20. Branford White CJ. Molecular organization of heparan sulphate proteoglycan from human dentine. Arch Oral Bio11978;23:1141-4.
The Way It Was In Kingston, New Hampshire, in 1730 a hog owned by a Mr. Clough was "taken sick of a complaint in the throat and died." Mr. CIough butchered the hog and subsequently also died. By the fall of 1731, 802 children under the age of 10 were dead from airway obstruction caused by "extensive membranous inflammation of the throat." Epidemic diphtheria had arrived in New England. William Cornelius
