Uptake of sulfate in developing bovine dental pulp: a connective tissue model

JOURNAl- OF ENDODONTICS I VOL 5, NO 10, OCTOBER 1979

Uptake of sulfate in developing bovine dental pulp: a connective tissue model James L. Snyder, DDS; Jim R. Patten, DMD; Stewart D. Turner, PhD; Max O. Hutchins, Gordon W. Heath, PhD, Houston

PhD;

and

Pulp tissue was extracted f r o m fresh p e r m a n e n t m o l a r s of a b o v i n e calf. Slices of the p u l p tissue w e r e i n c u b a t e d in the p r e s e n c e of ~-~SO U p t a k e of sulfate was g r e a t e s t in the apical a r e a s a n d in the p u l p tissue of the m o s t e m b r y o n i c molar. In addition, the i n c o r p o r a t i o n of sulfate into acid m u c o p o l y s a c c h a r i d e s was g r e a t e s t in the m o s t e m b r y o n i c p u l p tissue. T h i s p r o c e d u r e m a y serve as a m o d e l for studying pulp development.

]'he acid mucopolysaccharides, glycosaminoglycans (GAGs), are important biochemical components of dental pulp and its precursor, the dental papilla. IAnde' has identified specific GAGs from the pulp tissue of several species. Several GAGs, such as chondroitin sulfate and hyaluronic acid, were identitied, and their distribution was highly dependent on the stage of development of the dental pulp. These results were in agreement with histologic data previously reported by Fullmer.'-' Linde' and E m b e r y ' examined the in vivo uptake of :':'SO, as a method for studying sulfated glycosaminoglycan metabolism in pulp from the rat incisor. They reported that :";SO, was rapidly incorporated into :':'Slabeled GAGs in the ground substance of erupting teeth of rats. However, a review of the literature showed no studies examining the in vitro uptake of ' ' S O , by pulp tissue collected from different teeth in the same arch as well as difl'erent

anatomical areas of the pulp. Possible variation in :':'SOd uptake by different areas of pulp tissue is indicated by the variation in metabolic rate reported by Fisher' and Fisher and Schwabe? They h a v e shown that consumption of oxygen by bovine and rat pulp differs with respect to the region of the tooth from which the pulp was collected. The purpose of the current study is to identify the area of the pulp that maximally incorporates ....SO, into the sulfated GAGs in vitro. These data will be useful in developing a model system to study the effects of various hormones and medicaments on the growth and the development of dental pulp.

MATERIALS A N D METHODS Mandibles* were collected from slaughtered 600- to 900-1b calves. The mandibles were packed in ice, transported to the laboratory, and radiographs were taken to evaluate

dental age (Fig 1). Four mandibles were selected and fractured at the ramus. The following molars were collected from the cheek: the tirst erupted molar (MI), the second unerupted molar (MII), and the third embryonic molar (Mill). Dental pulp was collected and placed in cold Krebs balanced salt solution (BSS). The dental pulp was prepared for incubation under a tissue culture hood equipped with an ultraviolet light. The tissue was sectioned into apical, middle, and occlusal divisions (Fig 2) and sliced with a StadieRiggs knife into small pieces.' Each piece of pulpal tissue was added to a sterilized, preweighted, miniature, scintillation vial. These vials contained 2.0 ml of Eagle's medium (pH, 7.4) with gentamicin (50 m g / liter). The pulp tissue and media were preincubated for an hour in a Dubnoff shaker at 37 C. At the end of the preincubation period, 10 /.tCi of :';SO., carrier-free Na~SO, was 305

Fig 2-1solated pulp fiom MII (A ) and MHI (B) indicating approximate apical, middle, and occlusal thirds.

Fig 1-Radiographs of bovine mandible showing erupted first molar (MI), partially erupted second molar ( MII), and unerupted third molar (MHI).

diluted in 0.5 ml of buffered Krcbs BSS. The vials then were incubated for 18 hours at 37 C with gentle shaking. After incubation, the procedure of Herbal '~was used to interrupt further uptake of :;:'SO, into the dental pulp. The incubation medium was removed and the pulp washed with distilled water. Two milliliters of cold saturated Na2SO, was added to "quench" the uptake of :'"SO,. Another incubation of six hours at 37 C was conducted in the saturated sulfate solution. The tissue then was rewashed with water. In some experiments, a small section of the pulp >,'as taken from each vial, numbered, weighed, and frozen. The '"SO, uptake in synthesized glycosaminoglycans was determined in this tissue. The vials with the remaining pulp were placed in a drying oven at 80 C for three hours. After they were dried, the vials were weighed to the nearest milligram One ml of 5.25% sodium hypochlorite was added to each vial, and the pulp was digested for 24 hours. After digestion, 6.0 ml of Aquasolt liquid scintillation fluid was added to each vial. The vials were counted in a Packard Tri-Carb liquid scintillation counter.{ The average counts per minute/milligram of '"SO~ were cal306

culated for each pulpal site, for each tooth collected, and for each calf mandible reported in this work. An analysis of variance and t test were performed on these data. The. procedure of Di Ferrante" was used to determine the amount of :'"SO~ in the GAGs found in the bovine dental pulp. The frozen dental pulp sections were thawed and digested in a papain solution. The solution contained 50 mm cysteine-HCl (pH, 6.5). The pulp was digested for six hours at 65 C in a water bath. The digested tissue was dialvzed overnight and centrifuged for 30 minutes at 3,000 g. Heparin (0.5 rag) was added to the supernatam as a carrier and a 1% cetylpyridinium chloride (CPC) solution was added with drops until no further precipitate was formed. The precipitate was dissolved in ethanol, and Aquasol was added. The samples were counted and expressed as c p m / mg of wet pulp tissue. Pieces of pulp tissue from the occlusal, middle, and apical sections of MI, MI[, and MIII were fixed in buffered formaldehyde for histologic examination. This tissue was embedded in paraffin and sectioned and stained with hematoxylin and eosin according to standard, routine procedures. RESULTS The :':'SO, uptake ( c p m / m g of dry tissue) by incubated bovine dental pulp is shown in Table 1. Bovine molars MI, MII, and M I I I were collected from one calf mandible for each given experiment (A, B, and C).

The pulp was collected and divided into seven to 18 pieces, depending on the size of the tooth and its pulp. Incubated pulp tissue collected from the erupted first molar had significantly less ::'SO, in the tissues (2,541 e p m / m g ) than pulp tissue collected from the calcified unerupted second molar (6,798 crop/rag). The calculated mean v'SO, uptake (11,041 cprn/mg) of incubated pulp tissue collected from the embryonic third molars was significantly greater (P < .05) than the specific activity of ....SO, ( c p m / m g ) in pulp tissue collected from M[ or MII. The uptake of ....SO~ in three different regions of pulp tissue is shown in Table 2. These data reflect the :~"SO, uptake in the occlusal, middle, and apical areas of the dental pulp collected from the three permanent molars. An analysis of variance on the mean total '"SO, in the occlusal region of MI, MII, and MIII (4,278 c p m / m g ) was not significantly different from the specific activity measured in the middle region (5,711 cpm/mg). However, the amount of :';S(), in the occlusal area was significantly less (P < .05) than the mean specific activity of :~:'SO, in incubated apical pulp tissue (7,910 epm/mg). Incorporation of :"SO, into glycosaminoglycans in incubated bovine denial pulp is shown in Table 3. The pulp tissue samples were analyzed for each experiment and the specific activity of :'"SO, ranged from 18.6 to 69.8 c p m / m g of wet tissue for pulp taken from the erupted first molars. Pulp tissue collected from MII ranged from 46.2 to 74.8 c p m / m g in

JOURNAL OF ENDODONTICS VOL 5, NO 10, OCTOBER 1979

Table 1 9 Uptake of '~SO, in vitro in bovine dental pulp.

Mandibular teeth

Experiment

MI (5)*

A B C

MII (5)

A B C

MIll (5)

A B C

Total pulp pieces 9 18 18 45 9 18 18 45 9 8 7 24

~"SO, cpm/mg dry tissue 1020 1673 4170 2541 3179 4905 10,500 6798 8192 7146 19.156 11,041

_+ 248] _+ 127 _+ 452 _+ 279~ _+ 552 _+ 472 _+ 825 _-4- 606~ + 2251 + 950 _+ 2969 _+ 16163~

*'lt~tal teeth collectcd. 'l'Mcan • S E

~1' <..O5

Table 2 * Uptake of ~SO, in vitro in different pulp regions.

Mandibular teeth MI (5)* MII (5) MIII (5) Total

Occlusal cpm/mg

Pulpal regions Middle c p m / m g

2966 5462 4599 4278

1973 6180 11158 5711

_+ 555 (15)]. _+ 953 (15) +_ 1142 (15) -2_ 521a (36)

_+ 300 (15) _+ 1170 (15) _+ 2161 (09) _+ 870" "(39)

* l'otal teeth collected+ +Mean + SE (no. ol pulp pieces), Valuer, with the same letter subscript

are not

Apical cpm/mg 2684 8752 15219 7910

+_ 546 (15) _+ 871 (15) _+ 3028 (09) _+ 1097b (39)

si~nilicantly different

Table 3 9 Incorporation of ~'SO, into glycosaminoglycans in incubated bovine dental pulp.

Mandibular teeth

Experiment

MI

B C D

MII

B C D

Mill

No. of samples 3 3 3 9 3 3 3 9

B

3

C D

3 3 9

':'SO ~ uptake in GAGS (cpm/mg wet tissue) 35.0 18.6 69.8 41.3 + 13.8" 46.2 74.8 67.0 62.9 _+ 20.9 151.0 283.6 167.3 204.0 _+ 26.1t

"Mean • SE. +P-:: 115,

comparison with a range of 151 to 283.6 c p m / m g for the e m b r y o n i c pulp collected from the third molar. There were significantly more :~:'SO,labeled G A G s in the i n c u b a t e d p u l p tissue collected from the third molars

than in the p u l p tissue o b t a i n e d from M I a n d MII. T h e organic u p t a k e of :t;SO, by p u l p tissue collected from M I a n d M I I was not significantly difFerent. Histologic e x a m i n a t i o n s u p p o r t e d

the results of :~:'SO, u p t a k e in d e n t a l pulp. Pulp tissue from the third m o l a r was cellular (Fig 3), and the g r o u n d substance showed sparse fine collagen fibers. T h e p u l p tissue from M I I consisted of a reduced density of cells a n d a greater a m o u n t of g r o u n d substance a n d fibrous material. P u l p tissue from M I was even less cellular a n d much more fibrous. T h e r e was a g r a d a t i o n of decreased cellular density from M I I I through M I and from the apical to occlusal third of each. T h e apical third of M I I a n d especially of M I I I consisted of a large percentage of cells with little cytoplasm a n d large, vesicular nuclei indicative of undifferentiated cells. Progressive differentiation into mature cells was evident from M I I I to M I tissue as well as from the apical to occlusal third of each tooth. T h e r e was some variation in m a t u r i t y a n d n u m b e r of cells in different parts of the tissue taken from the i n d i c a t e d areas a n d teeth, but the a p p e a r a n c e of each location was easily distinguished. T h e histologic observations support the isotopic studies in that there is a cellular distinction between the more d e v e l o p e d first m o l a r a n d the e m b r y o n i c third molar. DISCUSSION

T h e connective tissue of the d e n t a l p u l p has m a n y actions a n d undergoes n u m e r o u s structural changes d u r i n g development. D u r i n g aging, in the p u l p as in other connective tissues, the q u a n t i t y of the g r o u n d substance a n d n u m b e r of cells decrease whereas collagen fibers increase. '~ After the d e n t a l p u l p has been injured, it undergoes structural changes similar to those that occur d u r i n g aging. T h e r e is proliferation of connective tissue ~ a n d formation of r e p a r a t i v e dentinY In some 307

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Fig 3-Pulp tissue from ca(f tooth (H & E, orig mag X ]00). A, first molar, occlusal region. Mostly mature fibroblasts wzth dark, dense nuclei and abundant collagenous .fibers. B, .first molar, apical re~ion. Fibroblasts have larger, less dense nuclei, indicating younger, more actwe cells. Scattered collagenousfibers. C, third molar, occlusal region. Mixture of mature.fibroblasts (dark, dense nuclei), young, active fbroblasts (larger, less dense nuclei) and perhaps some und!ff~erentiatedmesenchymal cells (largest, most vesicular nuclei). Moderate numbers of collagenous.fibers. D, third molar, apical region. Most cells are crowded, undifl~entiated mesenchymal cells with large, vesicular nuclei. Few fine collagenousfibers have firmed at this time.

instances, injured pulp may undergo dystrophic calcification, internal resorption, and alteration in apical closure. To understand the metabolic changes that occur in the dental pulp 308

during aging or injury, it is important to examine the biochemical components of the dental pulp. Embery > examined the distribution of GAGs in the pulp of permanent human molars and premolars and

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found chondroitin-4- and chondroitin-6-sulfate to be the major GAGs. Orlowski" has analyzed porcine and bovine dental pulp for the different proportions of collagen, glycoproteins, and acid mucopolysaccharides. He reported that the principal GAGs are hyaluronic acid and a sulfated component, chondroitin sulfate. These macromolecules are believed to have a profound influence on the organization of the extracellular components of the dental pulp. Radioactive :~:'S()4 has been used to study the synthesis of the sulfated GAGs in the dental pulp. ':~v-' Consequently, uptake of a:'SO, in pulpal tissue in vivo is a useful model for investigating the metabolism of the connective tissue ground substance. Before use of labeled sulfate as an indicator of GAGs synthesis in dental pulp, other investigators "~:' had used this procedure for measuring GAGs synthesis in connective tissue from other species or other anatomical locations. Herbal '; had previously observed that pieces of costal cartilage from the osteochondral junction had incorporated two to four times as much :':'SO, as the remaining homogenous parts of costal cartilage. The results of the current study also indicate that there are statistically significant differences in the metabolic uptake of:~"SO, in vitro in bovine pulp tissue with regard to the site within a tooth and with regard to the maturation of teeth. Fisher 4 previously studied metabolism of rat and bovine pulp tissue; his study showed progressively higher oxygen consumption rates from incisal to basal thirds and in peripheral vs central portions of bovine pulp. In another study, Fisher and Schwabe" observed that embryonic bovine pulp tissue had greater oxygen uptake than more

JOURNAL OF ENDODONTICS ' VOL 5, NO 10, OCTOBER 1979

mature tissue. These results are metabolically similar to the results observed in the current study that showed :';SO, uptake was greater in the apical section of the dental pulp as well as in the most e m b r y o n i c cheek molars. In addition, the organic incorporation of :':'SO, into GAGs was significantly higher in the embryonic third molars t h a n in the pulpal tissue of the differentiated first a n d second molars. This difference in uptake of :':'SO~ by dental pulp may be caused by variation in the a n i m a l ' s age, sex, or breed. However, it could be caused by significant changes in the synthesis of acid mucopolysaccharides, the metabolic turnover of these molecules in older teeth, or differences in the types of cells present. This is supported by our observation that n u m e r o u s undifferentiated cells were observed histologically in the apical section of the third cheek molars, whereas fewer undifferentiated cells were observed in the first a n d second molars. U i t t o " i n c u b a t e d dental pulp collected from h u m a n s a n d rabbits to study collagen m e t a b o l i s m in vitro. He found this procedure useful in e x a m i n i n g the effects of cortisol and antibiotics on collagen a n d protein synthesis in pulpal tissue. T h e primary purpose of this current investigation was to develop a similar connective tissue model with use of bovine dental pulp. Bovine dental pulp, easily o b t a i n e d at a low cost and available in large quantities, provides a model with a wide range of morphological development. Further studies with this tissue could explore n u m e r o u s therapeutic possibilities for h o r m o n a l control of normal physiologic processes that occur d u r i n g development, aging, and injury' of the dental pulp.

CONCLUSIONS T h e results of this study indicate that: - B o v i n e pulp tissue can be used to study the metabolism of the sulfated glycosaminoglycans. - T h e advantages of using this model system to evaluate biochemical c o m p o n e n t s of the pulp are tissue size, availability, and morphological variation. - P u l p tissue collected primarily from the noncalcified apical third of the molars takes up significantly more :~"SO., than the occlusal pulp tissue, a n d this :::'SO, is incorporated into sulfated glycosaminoglycans, especially by pulp tissue collected from the e m b r y o n i c third molars. - B e c a u s e of variation in :';SO, uptake between animals, control tissue for any experimental study should come from teeth on the same mandible. - T h i s procedure could serve as a connective tissue model for s t u d y i n g biological changes in d e n t a l pulp that occur in response to injury, medicaments, or hormones. *Blue Ribbon Packing Co., Houston. "~New England Nuclear, Boston. ~Packard Instrument Co., Inc., Downers Grove, Ill. This article was abstracted from a master's thesis submitted by Dr. J. Snyder to the Postgraduate School of Dentistry, University of Texas Health Science Center at Houston, Dental Branch. This study was partially supported by the American Associationof Endodontics Endowment and Memorial Foundation. The authors thank Mrs. R. Jenkins, Mrs. N. Steward, and Mr. V. Williams for their technical assistance. Dr. Snyder was a graduate student in endodontics at the University of Texas Dental Branch at Houston and is currently stationed

at Lackland Air Force Base in San Antonio. Dr. Patten is associate professor of endodontics and physiology. Dr. Hutchins is professor and chairman of physiology, and Dr. Heath is professor of mieroanatomy at the Universityof Texas Dental Branch at Houston. Requests for reprints should be directed to Dr. Jim Patten, Department of Endodontics, University of Texas Dental Branch, PO Box 20068, Houston, 77025.

References

1. Linde. A. Glycosaminogtycans of the dental pulp. A biochemical study. Stand J Dent Res 81:177-201, 1973. 2. Fullmer, H.M. Histochemical studies of the acid mucopolysaccharides. Oral Surg 33:976-82, 1972. 3. Embery,G. The isolation of chondroitin 4-(:'"S) sulphate from the molar teeth of young rats receiving sodium ('"S) sulphate. Calcif Tissue Res 14:59-65, 1974. 4. Fisher, A.K. Respiratory variation within the normal dental pulp. J Dent Res 46:454428, 1967. 5. Fisher,A.K., and Schwabe, C. The endogenous respirator3" quotient bovine dental pulp. J Dent Res 40:346-351, 1961. 6. Herbai, G. Incorporation and disappearance of sulfate in different regions of mouse and rat costal cartilage in vivo and in vitro. Acta Physiol Scand 79:541-551, 1970. 7. Di Ferrante, N. Personal communication. 8. Sayegh, F.S. Healing of the traumatized dental pulp. J Dent Res 46:1036-1(143, 1967. 9. Philippas, G.G., and Applebaum, E. Age factor in secondary dentin formation. J Dent Res 45:778-789. 1966. 10. Ember2,', G. Glycosaminoglycans of human dental pulp..j" Biol Bucca[e 4:229-236, 1976. 11. Orlowski, W.A. Analysis of collagen, glycoproteins and acid mucopolysaccharides in the bovine and porcine dental pulp. Arch Oral Biol 19:255-258, 1974. 12. Blumen, G., and Merzel, J. Autoradiographic study with ('"S)-sodium sulphate of loss of sulphated glycosaminoglycansduring ameologenesis in the guinea pig. Arch Oral Biol 21:513-521, 1976. 13. Toole, B.P., and Gross,J. The extraccllular matrix of the regenerating new limb synthesis and removal of hyaluronate prior to differentiation. Dev Biol 25:57-77, 1971. 14. Uitto, v.J. Collagen biosynthesis of pulp. Proc Finn Dent Soc 72 (suppl 1-3): 1, 1976. 309