Archs oral Bid. Vol.37, No. 5, pp. 323-330, Printed in Great Britain. All rights reserved
1992 Copyright
GLYCOSAMINOGLYCAN PROTEOGLYCANS
0
0003-9969/92 $5.00 + 0.00 1992 Pergamon Press Ltd
PATTERNS IN GINGIVAL OF RAT WITH AGE
M. WEINSTEIN,Y. H. LIAU, A. SLOMIANY and B. L. SLOMIANY* Research Center, New Jersey Dental School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103-2400, U.S.A. (Accepted 28 November 1991)
Summary-Among the potential biochemical indices that are closely associated with craniofacial development are the proteoglycans. Gingival segments from the palate of 4-,6-,8-,12- and 18-week-old rats were incubated for 4 h in medium containing [‘HI-glucosamine and [“S]-Na,SO,, and subjected to proteoglycan isolation and glycosaminoglycan analysis. Two distinct proteoglycan fractions differing in the degree of sulphation were obtained by ion-exchange chromatography. The incorporation of both labels in the undersulphated fraction increased with age; there was a pronounced decrease with age in the sulphated proteoglycan fraction. The undersulphated proteoglycans showed an age-dependent decrease in hyaluronic acid, and increase in dermatan sulphate and chondroitin 4- and 6-sulphates. Gel filtration of the sulphated proteoglycan fraction yielded high and low molecular-weight proteoglycans, the glycosaminoglycans of which were particularly rich (61-76%) in dermatan sulphate. Smaller quantities of chondroitin 4- and 6-sulphates, and heparan sulphate were also present. All glycosaminoglycans showed a decrease in content with age. The findings suggest a possible correlation between gingival proteoglycan/glycosaminoglycan patterns and development. Key words: gingiva, proteoglycan, glycosaminoglycan, patterns, developmental changes, rat.
INTRODUCTION In orthodontics the accurate measurement and prediction of craniofacial growth is important, as small differences can profoundly affect the type and success of treatment. The current, standard technique for assessing craniofacial growth and maturation is the hand wrist radiograph (Greulich and Pyle, 1959; Houston, Miller and Tanner, 1979). However, this technique suffers from its reliance on a noncraniofacial structure and the use of radiation. Hence, a more accurate measurement or technique that avoids radiation and allows direct determination of craniofacial age and growth would be of great advantage to orthodontics. The proteoglycans are among the potential biochemical indices that appear to be closely associated with craniofacial growth and development (Madsen et al., 1983; Bartold, 1987). Studies on epiphyseal cartilage indicate a progressive decrease in proteoglycans with age (Kimata et al., 1974) and there appears to be a sharp decline and change with age in the synthesis and composition of the glycosaminoglycans of auricular cartilage (Madsen et al., 1983). A clear relationship has been established between gingival development and its glycosaminoglycans, with dermatan sulphate and chondroitin 4-sulphate markedly decreasing with age (Sakamoto, Okamoto and Okuda, 1978). We have now used palatal gingiva of growing rats at 4, 6, 8, 12 and 18 weeks of age to investigate developmental patterns of proteoglycans and glycosaminoglycans. *To whom all correspondence should be addressed.
MATERIALSAND
METHODS
Materials
Radioactive precursors, [35S]-Na,S0, (50 mCi/ mmol) and [3H]-glucosamine HCl (30 Ci/mmol) were from New England Nuclear, Boston, MA, U.S.A. Scintillation solutions, Optifluor and Insta Gel, were from Packard Instrument Co., Downers Grove, IL, U.S.A. Joklick modified minimal essential medium was obtained from Grand Island Biochemical Corp., Grand Island, NY, U.S.A, chondroitinase ABC and AC, chondro 4- and 6-sulphatases, heparinase III, keratinase and papain were from Sigma Chemical Co., St Louis, MO, U.S.A. Streptomyces hyaluronidase from Calbiochem, San Diego, CA, U.S.A. DEAE-Sephacel from Pharmacia, Piscataway, NJ, U.S.A. and Bio-Gel P-30, Bio-Gel A-l 5 and Bio-Gel A-50 from Bio-Rad Laboratories, Rockville Center, NY, U.S.A. Bicinchoninic Acid, protein assay kit was purchased from Pierce, Rockford, IL, U.S.A., male Sprague-Dawley rats (3, 5, 7, 11 and 17 weeks old) were obtained from Taconic Farms, Germantown, NY, U.S.A. Tissue collection
Twenty rats were used for each age-group experiment. They were kept in the animal house and their birthdate and weight recorded before they were killed with a solution of 25% urethane (0.5 ml/100 g body weight) given by intraperiotoneal injection to first induce anaesthesia. This procedure allowed time for careful dissection of the gingival specimens without affecting the tissue viability. The attached gingiva overlying the palate in the region between the molars 323
M. WEINSTEIN el al.
324
was gently reflected, incised with a scalpel, and lifted with a small periosteal elevator (Plate Fig. 1). Tissue culture
For the tissue culture, the specimens from each age group (4, 6, 8, 12 and 18 weeks) were immediately placed in a test tube containing ice-cold saline. The tissue was first rinsed with phosphate-buffered saline (0.05 M phosphate buffer-O.1 M NaCl, pH 7.2) and preincubated with 10 ml of modified minimal essential medium under 95% 0, and 5% CO, for 30 min. After separating from the medium, the tissue was resuspended in 5 ml of the modified medium containing [35S]-NazS0, and [3H]-glucosamine and incubated at 37°C for 4 h. Extraction
of proteoglycans
After culture, samples were rinsed twice with phosphate-buffered saline, centrifuged for 5 min at 10,000 rpm, and the wet weight of each sample was recorded. The tissue from each age group was placed in a separate tube containing guanidine extraction mixture consisting of 4 M guanidine HCl, 0.5 mM N-ethylmaleimide, 5 mM 3-chlolamidopropyl dimethyl amino- 1-propane sulphonate, 0.6 mM 6-aminohexanoic acetate, 50 mM disodium EDTA and 50mM sodium acetate, pH 5.8. The mixture was homogenized in a Tekmar Tissuemizer and left at room temperature with constant stirring for 16 h (Tian et al., 1986). The extract was then dialysed against distilled water, lyophilized and reconstituted in 2 ml of 6 M urea. The extracts were then subjected to gel filtration on a Bio-Gel P-30 column (0.7 x 18 cm) to separate radiolabelled material from free tracers, and the excluded fractions containing proteoglycans were applied to DEAESephacel column (1.5 x 15 cm), which before application was equilibrated and washed with 2 M NaCl in 6 M urea followed by 0.10 M NaCl in 6 M urea. The column was eluted stepwise with 0. I O-2 M NaCl in 6 M urea. Each fraction was monitored for radiolabel, dialysed against distilled water, and lyophilized. The radiolabelled fractions from the ion-exchange chromatography were then subjected to gel filtration on a Bio-Gel A-50 column (2 x 170 cm) preequilibrated with 6 M urea. The eluted fractions were monitored for proteins at 280nm and for radioactivity, and the high and low molecular-weight fractions were separately pooled, dialysed and lyophilized. Each fraction was then analysed for the total incorporation of radioactive labels by scintillation spectrophotometry and for protein content by bicinchoninic acid (Smith et al., 1985). Characterization and analysis of glycosaminoglycans
The high and low molecular-weight proteoglycan fractions were incubated (at 5 mg/ml) in 0.1 M sodium acetate containing I mM EDTA and 5 mM
Plate Fig. 1. The palate
cysteine, pH 5.5, at 60°C for 18 h. To each incubation mixture, 50% trichloracetic acid was added to a final concentration of 7% and the trichloracetic acidsoluble fraction was then dialysed against distilled water and lyophilized. The resulting powder was reconstituted with distilled water and used for the analysis of glycosaminoglycans. For the analysis of dermatan sulphate and chondroitin 4- and 6-sulphates, the samples were digested with chondroitinase ABC or AC to yield unsaturated disaccharides (Saito, Yamagata and Suzuki, 1968) and these were separated by thin-layer chromatography (Wasserman, Ber and Allalouf, 1977). The amount of dermatan sulphate and chondroitin 4- and 6-sulphates was calculated from the radioactivity of the resulting ADi4-S or ADiBS disaccharides. The amount of non-sulphated chondroitin was estimated from the radioactivity of ADIOS. To confirm the amount of each type of chondroitin sulphate, the samples were also digested with the combinations of chondroitinase and chondrosulphatase as follows: (1) chondroitinase ABC plus chondro-4-sulphatase; (2) chondroitinase AC plus chondro-Csulphatase and (3) chondroitinase ABC plus chondro-6-sulphatase. The resulting inorganic sulphate was precipitated as [35S]-BaS04 and its radioactivity measured. The amounts of chondroitin 4- and 6-sulphates were determined directly from the combination of steps 2 and 3, and the amount of dermatan sulphate was calculated by subtracting step 2 from 1. For the determination of heparan sulphate and keratan sulphate, each sample was incubated separately with heparinase and keratanase, the digests were fractionated on Bio-Gel P-30 columns and the amount of each glycosaminoglycan was determined from the radioactivity of the included fraction (Nakano and Scott, 1989). Hyaluronic acid was assayed by digestion with Streptomyces hyaluronidase (Brandt, Palonski and Perricone, 1976).
RESULTS
The DEAE-Sephacel chromatographic patterns of 4M-guanidine extracts of rat gingival proteoglycans are shown in Text Fig. 2. Two distinct peaks were obtained, one at 0.25 M NaCl (fraction I), and the other at 0.55 M NaCl (fraction II). Both fractions showed the presence of radiolabel and gave a positive reaction for uranic acid (Bitter and Muir, 1962), thus indicating the presence of proteoglycans. The ratio of incorporation of [3H]-glucosamine (measurement of glycosylation) to that of [3SS]-sulphate (measurement of sulphation) was, however, much higher in fraction I than in fraction II, (10 : 1 versus 2 : 1). This indicated a lesser degree of proteoglycan sulphation in fraction I than II. The total radiolabel incorporations for each fraction are given in Table 1. Although fraction II was the major fraction in all age groups, the proportion of the two fractions differed for each
I
of rat as used for gingival
tissue sampling.
Gingival
proteoglycans
Plate
1
325
M. WEINSTEIN et al.
326
---4 WEEKS 6 WEEKS . . . . . . . . . 8 WEEKS
(A)
_
I _,_ 1-
-
- -
(A)
A
(2 WEEKS 18 WEEKS
II
,_ In
i
8 ----
0.2
0.4
0.6
0.8
0.4
0.6
0.8
I
NaCl (Ml
Fig. 2. DEAE-Sephacel chromatographic profiles of 4Mguanidine chloride proteoglycan extracts of gingival tissue from rats of various age groups: (A) incorporation of [‘HI-glucosamine; (B) incorporation of [?C+sulphate. The material was pooled as indicated. (I) Fraction I. (II) Fraction II. Elution profiles represent typical patterns obtained for samples from each group.
age group. In fraction I, the incorporation of [35S]sulphate an [3H]-glucosamine increased with advance in age, while the reverse was observed in proteoglycan fraction II where the incorporation of both labels significantly decreased with age. Fraction II from the DEAE-Sephacyl column was further examined on Bio-Gel A-50, while fraction I,
25
FRACTION
4 WEEKS
6 WEEKS . . . . . . . . . 8 WEEKS -
0
4
-
- -
(2 WEEKS 18 WEEKS
50
75
101
NUMBER
Fig. 3. Bio-Gel A-50 column chromatography of gingival proteoglycan fraction II from DEAESephacel. Samples of proteoglycans from different age groups were applied separately to a column (0.9 x 120 cm) and eluted with 6 M urea. Fractions of 1.2 ml were collected and monitored for [‘HIglucosamine and [%I-sulphate by scintillation spectrometry. (A) Fraction IIA; (B) fraction IIB. Elution profiles are typical patterns from each age group.
because of the limited amounts, was analysed for glycosaminoglycans directly. Representative patterns on A-50 gel chromatography of the gingival proteoglycans in fraction II are shown in Text Fig. 3. Each age group had two distinct proteoglycan populations. The excluded fraction, fraction IIA, contained the
Gingival proteoglycans
327
L
w
_ (B)
q
4 WEEKS 6 WEEKS
-
l-
TOTAL
HA
GAG
Fig. 4. Distribution of hyaluronic acid (HA) and chondroitinase ABC-digestible glycosaminoglycans (GAG) in proteoglycan fraction 1 (Text Fig. 2) of rat gingiva. Panel A, incorporation of [3H]-glucosamine; panel B, incorporation of [35S]-sulphate. Bars represent the means + SD of values obtained for each age group.
Table 1. Distribution of [“%I-sulphate and [3H]-glucosamine incorporation in gingival proteoglycan fractions I and II from a DEAE-Sephacel column dis/min/g wet weight Age (weeks) 4 6 8 12 18 4 6 8 12 18
Fraction I
Fraction II
[35S]-Sulphate 7800 f 840 194,000 f 18,400 8400 & 860 131,000 + 13,050 12,lOOk 1180 87,700 + 9100 14,200 It 1500 48,300 k 5000 18,600 k 1820 32,000 k 3500 [3H]-Glucosamine 72,000 * 7900 82,000 + 8800 124,000 k 12,000 146,000 + 14,100 156,000 k 15,400
369,000 k 35,000 249,000 + 26,100 167,000 f 16,200 124,000 f 12,300 104,OOOf 11,150
Each value is the mean f SD of triplicate analyses made on each sample.
high-molecular weight (M, > 2 x 106) proteoglycan population, while the included fraction, Fraction IIB, represented the lower-molecular weight (M, < 2 x 106) proteoglycan population. Both populations were present in similar proportions in all age groups. The glycosaminoglycan analyses, made on the proteoglycan fraction I from DEAE-Sephacyl and on the two different molecular-weight proteoglycan populations (IIA and IIB) obtained from fraction II after Bio-Gel A-50 column chromatography, are summarized in Text Figs 4 and 5. The glycosaminoglycans in proteoglycan fraction I showed an agedependent decrease in hyaluronic acid content, and a considerable increase in dermatan sulphate and chondroitin 4- and 6-sulphates. At 4 weeks of age the hyaluronic acid accounted for 46.2% of glycosaminoglycans and dermatan sulphate and chondroitin 4and 6-sulphates for 56.6%; in the l&weeks-old group, the hyaluronic acid amounted to 12.2% of
M. WEINSTEIN et al.
328
m 4 WEEKS
2000
a6 m
.c_ 2 h ? .5 E \ 9
D Id 2
WEEKS 6 WEEKS
tDI2 WEEKS
q l8WEEKS
1000
0
5 5000 : 3 0 ; 2500
0
J
TOTAL
DS
c 4-s
c 6-S
HS
I (B)
J TOTAL
DS
c 4-s
C 6-S
HS
Fig. 5. Distribution of [‘HI-glucosamine and [‘5S]-sulphate in glycosaminoglycans of the high molecularweight (fraction IIA) and the low molecular-weight (fraction IIB) proteoglycan populations derived from fraction II after column chromatography on Bio-Gel A-50 (Text Fig. 3). (A) Glycosaminoglycans of Fraction IIA. (B) Glycosaminoglycans of fraction IIB. Bars represent the means + SD of values obtained for each age group. DS, C4-S, C6-S, C6-S and HS denote dermatan sulphate, chondroitin Csulphate, chondroitin 6-sulphate and heparan sulphate, respectively.
glycosaminoglycans, and dermatan sulphate and chondroitin sulphate 4- and 6-sulphates to 86.4% (Text Fig. 4). The data on glycosaminoglycans of the high moleeular-weight proteoglycans excluded from Bio-Gel A-SO, (fraction llA, Text Fig. 3), showed that most of the [35S]-sulphate and [3H]-glucosamine had heen incorporated into dermatan sulphate, followed by
chondroitin 4-sulphate, heparan sulphate, and chondroitin 6-sulphate. None of the age groups showed
the presence of keratan sulphate, and the pattern for each glycosaminoglycan detected remained essentially constant with age. In the 4-week age group, the dermatan sulphate accounted for 60.7% of glycosaminoglycans, heparan sulphate for 10.2%, chondroitin 4-sulphate for 20.1% and chondroitin
Gingival proteoglycans
329
6-sulphate for 3.0%; in the 18-week age group 56.1% of glycosaminoglycans were represented by dermatan sulphate, 20.3% by chondroitin 4sulphate, 10.8% by heparan sulphate, and 3.1% by chondroitin 6-sulphate. Even higher proportions of dermatan sulphate were found among the glycosaminoglycans of the proteoglycan fraction IIB that was included on Bio-Gel A-SO (Text Fig. 3). The glycosaminoglycans of this fraction contained 73-76% of dermatan sulphate, 8-9% chondroitin 4-sulphate, 4.2-4.7% chondroitin 6-sulphate, and 3.54.4% heparan sulphate. However, in comparison to fraction IIA, the glycosaminoglycans of fraction IIB contained about 50% less heparan sulphate (Text Fig. 5).
ageing (Sakamoto et al., 1978). We show that in rat gingival tissue, with increasing age, there is a definite decrease in the synthesis of hyaluronic acid and a pronounced fall in dermatan sulphate, chondroitin 4and 6-sulphates, and heparan sulphate. These data thus lend further support to the notion that the sulphation patterns of glycosaminoglycans and proteoglycans in gingiva may correlate more closely with the stage of growth and development than the currently used indices for the prediction of craniofacial growth.
DISCUSSION
REFERENCES
Our findings clearly show a change in the proteoglycan patterns of rat gingival tissue with age. Although all age groups showed, on DEAESephacel, the presence of undersulphated and sulphated proteoglycan fractions, the undersulphated fraction increased significantly with age, while the sulphated fraction showed a marked decrease. This closely matches the findings of Tian et al. (1986): for the proteoglycans of bone matrix, under similar conditions, they found a small fraction of [3sS]labelled material eluted with 0.28 M NaCl and a major fraction with 0.68 M NaCl; and, as in our study, after gel chromatography, they found two peaks for two different proteoglycan populations. The earlier work on the identification and quantitation of the glycosaminoglycans in gingiva has produced a variety of findings. Only chondroitin 4- and 6-sulphates were identified in digests of human gingiva by Ciancio and Mather (1971). Subsequently, dermatan sulphate, chondroitin 4-sulphate, hyaluronic acid and heparan sulphate were detected in porcine gingiva, but not chondroitin 6-sulphate (Hiramatsu, Abe and Minami, 1978). Other studies with human gingiva have shown variable amounts of dermatan sulphate and chondroitin 4- and 6-sulphate (Bartoid, 1987). We found that the major glycosaminoglycan of rat gingiva is dermatan sulphate, with chondroitin 4- and 6-sulphates, hyaluronic acid, and heparan sulphate also present, but keratan sulphate undetected. These findings are thus in agreement with those Bartold et al. (1981, 1987) and of Dahllof et af., (1986). A progressive decrease in proteoglycan synthesis with age in the embryonic chick has been reported by Robinson and Dorfman (1969) and Kimata et al. (1974). Developmental or age-associated changes in glycosaminoglycans show a decrease in the ratio of chondroitin 4- and 6-sulphates in chick epiphyseal cartilage (Kimata et al., 1974; Robinson and Dorfman, 1979) and an increase in chondroitin 6sulphate in the auricular cartilage of the rabbit (Madsen et al., 1983). For gingival tissue, investigations on material from cows ranging in age from 2 weeks to 1Oyr revealed a rapid decrease in glycosaminoglycans during the first 3 h of development followed further by less evident changes with
Bartold P. (1987) Proteoglycans of the periodontium: structure, role and function. J. periodont. Res. 22, 431-444. Bartold P., Wiebkin 0. and Thonard J. (1981) Glycosaminoglycans of human gingival epithelium and connective tissue. Connecl. Tiss. 9, 99-106. Bitter T. and Muir H. (1962) Determination of uranic acid using a carbozole reagent. Analyt. Biochem. 4, 330-334. Brandt K. D., Palonski M. J. and Perricone E. (1976) Aggregation of cartilage proteoglycans. II. Evidence for the presence of a hyaluronate-binding region on proteoglycans from osteoarthritic cartilage. Arfhrifis Rhem. 19, 1308-1314. Ciancio S. and Mather M. (1971) Acid mucopolysaccharides in gingivitis and periodontitis. J. periodont. Res. 6, 188-193. Dahllof G., Modeer T., Reinholt F., Wikstrom B. and Hjerpe A. (1986) Proteoglycans and glycosaminoglycans in phenytoin-induced gingival overgrowth. J. periodont. Res. 21, 13-21. Greulich W. and Pyle S. (1959) Radiographic Atlas of Skeletal Development of the Hand and Wrist, 2nd edn. Stamford University Press. Stanford, CA. Hiramatsu M.. Abe I. and Minami N. (1978) Acid mucopolysaccharides in porcine gingiva. J. ieriohont. Res. 13, 224-227. Houston N., Miller J. and Tanner J. (1979) Prediction of the timing of adolescent growth spurt from ossification events in hand-wrist films. >r. J. &ho. 6, 145-152. Kimata K.. Okavama M.. Oohira A. and Suzuki S. (1974) Heterogeneity of protedchondroitin sulfates produced by chondrocytes at different stages of cytodifferentiation. J. biol. Chem. 249, 16461653. Madsen K., Maskalewski S., Von Der Mark K. and Frieberg U. (1983) Synthesis of proteoglycans, collagen and elastin by culture of rabbit auricular chondrocytesRelation to age of the donor. Devl Biol. 96, 63-73. Nakano T. and Scott P. G. (1989) A quantitative chemical study of glycosaminoglycans in the articular disk of the bovine temporomandibular joint. Archs oral Biol. 34, 749-757.
Acknowledgement-This work was supported by Grant DE05666-I3 from the National Institute of Dental Research, National Institutes of Health.
Robinson H. and Dorfman A. (1969) The sulfation of chondroitin sulfate in embryonic chick cartilage epipyses. J. biol. Chem. 244, 348-352. Saito H., Yamagata T. and Suzuki S. (1968) Enzymatic methods for the determination of small quantities of isomeric chondroitin sulfates. J. Biol. Chem. 243, 15361542. Sakamoto N., Okamoto H. and Okuda K. (1978) Qualitative and quantitative analysis of bovine gingival glycosaminoglycans. Archs oral Biol. 23, 983-987.
330
M. WEINSTEIN et al.
Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J. and Klenk D. C. (1985) Measurement of protein using bicinchoninic acid. Analyf. Biochem. 150, 7685. Tian M., Yanagishita M., Hascall V. and Reddi A. (1986) Biosynthesis and fate of proteoglycans in cartilage
and bone during development and mineralization. Arch. Biochem. Biophys. 241, 221-232. Wasserman L., Ber A. and Allalouf D. (1977) Use of thin-layer chromatography in the separation of disaccharides resulting from digestion of chondroitin sulphates with chondroitinases. J. Chromatog. 36, 342-347.
1992 Copyright
GLYCOSAMINOGLYCAN PROTEOGLYCANS
0
0003-9969/92 $5.00 + 0.00 1992 Pergamon Press Ltd
PATTERNS IN GINGIVAL OF RAT WITH AGE
M. WEINSTEIN,Y. H. LIAU, A. SLOMIANY and B. L. SLOMIANY* Research Center, New Jersey Dental School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103-2400, U.S.A. (Accepted 28 November 1991)
Summary-Among the potential biochemical indices that are closely associated with craniofacial development are the proteoglycans. Gingival segments from the palate of 4-,6-,8-,12- and 18-week-old rats were incubated for 4 h in medium containing [‘HI-glucosamine and [“S]-Na,SO,, and subjected to proteoglycan isolation and glycosaminoglycan analysis. Two distinct proteoglycan fractions differing in the degree of sulphation were obtained by ion-exchange chromatography. The incorporation of both labels in the undersulphated fraction increased with age; there was a pronounced decrease with age in the sulphated proteoglycan fraction. The undersulphated proteoglycans showed an age-dependent decrease in hyaluronic acid, and increase in dermatan sulphate and chondroitin 4- and 6-sulphates. Gel filtration of the sulphated proteoglycan fraction yielded high and low molecular-weight proteoglycans, the glycosaminoglycans of which were particularly rich (61-76%) in dermatan sulphate. Smaller quantities of chondroitin 4- and 6-sulphates, and heparan sulphate were also present. All glycosaminoglycans showed a decrease in content with age. The findings suggest a possible correlation between gingival proteoglycan/glycosaminoglycan patterns and development. Key words: gingiva, proteoglycan, glycosaminoglycan, patterns, developmental changes, rat.
INTRODUCTION In orthodontics the accurate measurement and prediction of craniofacial growth is important, as small differences can profoundly affect the type and success of treatment. The current, standard technique for assessing craniofacial growth and maturation is the hand wrist radiograph (Greulich and Pyle, 1959; Houston, Miller and Tanner, 1979). However, this technique suffers from its reliance on a noncraniofacial structure and the use of radiation. Hence, a more accurate measurement or technique that avoids radiation and allows direct determination of craniofacial age and growth would be of great advantage to orthodontics. The proteoglycans are among the potential biochemical indices that appear to be closely associated with craniofacial growth and development (Madsen et al., 1983; Bartold, 1987). Studies on epiphyseal cartilage indicate a progressive decrease in proteoglycans with age (Kimata et al., 1974) and there appears to be a sharp decline and change with age in the synthesis and composition of the glycosaminoglycans of auricular cartilage (Madsen et al., 1983). A clear relationship has been established between gingival development and its glycosaminoglycans, with dermatan sulphate and chondroitin 4-sulphate markedly decreasing with age (Sakamoto, Okamoto and Okuda, 1978). We have now used palatal gingiva of growing rats at 4, 6, 8, 12 and 18 weeks of age to investigate developmental patterns of proteoglycans and glycosaminoglycans. *To whom all correspondence should be addressed.
MATERIALSAND
METHODS
Materials
Radioactive precursors, [35S]-Na,S0, (50 mCi/ mmol) and [3H]-glucosamine HCl (30 Ci/mmol) were from New England Nuclear, Boston, MA, U.S.A. Scintillation solutions, Optifluor and Insta Gel, were from Packard Instrument Co., Downers Grove, IL, U.S.A. Joklick modified minimal essential medium was obtained from Grand Island Biochemical Corp., Grand Island, NY, U.S.A, chondroitinase ABC and AC, chondro 4- and 6-sulphatases, heparinase III, keratinase and papain were from Sigma Chemical Co., St Louis, MO, U.S.A. Streptomyces hyaluronidase from Calbiochem, San Diego, CA, U.S.A. DEAE-Sephacel from Pharmacia, Piscataway, NJ, U.S.A. and Bio-Gel P-30, Bio-Gel A-l 5 and Bio-Gel A-50 from Bio-Rad Laboratories, Rockville Center, NY, U.S.A. Bicinchoninic Acid, protein assay kit was purchased from Pierce, Rockford, IL, U.S.A., male Sprague-Dawley rats (3, 5, 7, 11 and 17 weeks old) were obtained from Taconic Farms, Germantown, NY, U.S.A. Tissue collection
Twenty rats were used for each age-group experiment. They were kept in the animal house and their birthdate and weight recorded before they were killed with a solution of 25% urethane (0.5 ml/100 g body weight) given by intraperiotoneal injection to first induce anaesthesia. This procedure allowed time for careful dissection of the gingival specimens without affecting the tissue viability. The attached gingiva overlying the palate in the region between the molars 323
M. WEINSTEIN el al.
324
was gently reflected, incised with a scalpel, and lifted with a small periosteal elevator (Plate Fig. 1). Tissue culture
For the tissue culture, the specimens from each age group (4, 6, 8, 12 and 18 weeks) were immediately placed in a test tube containing ice-cold saline. The tissue was first rinsed with phosphate-buffered saline (0.05 M phosphate buffer-O.1 M NaCl, pH 7.2) and preincubated with 10 ml of modified minimal essential medium under 95% 0, and 5% CO, for 30 min. After separating from the medium, the tissue was resuspended in 5 ml of the modified medium containing [35S]-NazS0, and [3H]-glucosamine and incubated at 37°C for 4 h. Extraction
of proteoglycans
After culture, samples were rinsed twice with phosphate-buffered saline, centrifuged for 5 min at 10,000 rpm, and the wet weight of each sample was recorded. The tissue from each age group was placed in a separate tube containing guanidine extraction mixture consisting of 4 M guanidine HCl, 0.5 mM N-ethylmaleimide, 5 mM 3-chlolamidopropyl dimethyl amino- 1-propane sulphonate, 0.6 mM 6-aminohexanoic acetate, 50 mM disodium EDTA and 50mM sodium acetate, pH 5.8. The mixture was homogenized in a Tekmar Tissuemizer and left at room temperature with constant stirring for 16 h (Tian et al., 1986). The extract was then dialysed against distilled water, lyophilized and reconstituted in 2 ml of 6 M urea. The extracts were then subjected to gel filtration on a Bio-Gel P-30 column (0.7 x 18 cm) to separate radiolabelled material from free tracers, and the excluded fractions containing proteoglycans were applied to DEAESephacel column (1.5 x 15 cm), which before application was equilibrated and washed with 2 M NaCl in 6 M urea followed by 0.10 M NaCl in 6 M urea. The column was eluted stepwise with 0. I O-2 M NaCl in 6 M urea. Each fraction was monitored for radiolabel, dialysed against distilled water, and lyophilized. The radiolabelled fractions from the ion-exchange chromatography were then subjected to gel filtration on a Bio-Gel A-50 column (2 x 170 cm) preequilibrated with 6 M urea. The eluted fractions were monitored for proteins at 280nm and for radioactivity, and the high and low molecular-weight fractions were separately pooled, dialysed and lyophilized. Each fraction was then analysed for the total incorporation of radioactive labels by scintillation spectrophotometry and for protein content by bicinchoninic acid (Smith et al., 1985). Characterization and analysis of glycosaminoglycans
The high and low molecular-weight proteoglycan fractions were incubated (at 5 mg/ml) in 0.1 M sodium acetate containing I mM EDTA and 5 mM
Plate Fig. 1. The palate
cysteine, pH 5.5, at 60°C for 18 h. To each incubation mixture, 50% trichloracetic acid was added to a final concentration of 7% and the trichloracetic acidsoluble fraction was then dialysed against distilled water and lyophilized. The resulting powder was reconstituted with distilled water and used for the analysis of glycosaminoglycans. For the analysis of dermatan sulphate and chondroitin 4- and 6-sulphates, the samples were digested with chondroitinase ABC or AC to yield unsaturated disaccharides (Saito, Yamagata and Suzuki, 1968) and these were separated by thin-layer chromatography (Wasserman, Ber and Allalouf, 1977). The amount of dermatan sulphate and chondroitin 4- and 6-sulphates was calculated from the radioactivity of the resulting ADi4-S or ADiBS disaccharides. The amount of non-sulphated chondroitin was estimated from the radioactivity of ADIOS. To confirm the amount of each type of chondroitin sulphate, the samples were also digested with the combinations of chondroitinase and chondrosulphatase as follows: (1) chondroitinase ABC plus chondro-4-sulphatase; (2) chondroitinase AC plus chondro-Csulphatase and (3) chondroitinase ABC plus chondro-6-sulphatase. The resulting inorganic sulphate was precipitated as [35S]-BaS04 and its radioactivity measured. The amounts of chondroitin 4- and 6-sulphates were determined directly from the combination of steps 2 and 3, and the amount of dermatan sulphate was calculated by subtracting step 2 from 1. For the determination of heparan sulphate and keratan sulphate, each sample was incubated separately with heparinase and keratanase, the digests were fractionated on Bio-Gel P-30 columns and the amount of each glycosaminoglycan was determined from the radioactivity of the included fraction (Nakano and Scott, 1989). Hyaluronic acid was assayed by digestion with Streptomyces hyaluronidase (Brandt, Palonski and Perricone, 1976).
RESULTS
The DEAE-Sephacel chromatographic patterns of 4M-guanidine extracts of rat gingival proteoglycans are shown in Text Fig. 2. Two distinct peaks were obtained, one at 0.25 M NaCl (fraction I), and the other at 0.55 M NaCl (fraction II). Both fractions showed the presence of radiolabel and gave a positive reaction for uranic acid (Bitter and Muir, 1962), thus indicating the presence of proteoglycans. The ratio of incorporation of [3H]-glucosamine (measurement of glycosylation) to that of [3SS]-sulphate (measurement of sulphation) was, however, much higher in fraction I than in fraction II, (10 : 1 versus 2 : 1). This indicated a lesser degree of proteoglycan sulphation in fraction I than II. The total radiolabel incorporations for each fraction are given in Table 1. Although fraction II was the major fraction in all age groups, the proportion of the two fractions differed for each
I
of rat as used for gingival
tissue sampling.
Gingival
proteoglycans
Plate
1
325
M. WEINSTEIN et al.
326
---4 WEEKS 6 WEEKS . . . . . . . . . 8 WEEKS
(A)
_
I _,_ 1-
-
- -
(A)
A
(2 WEEKS 18 WEEKS
II
,_ In
i
8 ----
0.2
0.4
0.6
0.8
0.4
0.6
0.8
I
NaCl (Ml
Fig. 2. DEAE-Sephacel chromatographic profiles of 4Mguanidine chloride proteoglycan extracts of gingival tissue from rats of various age groups: (A) incorporation of [‘HI-glucosamine; (B) incorporation of [?C+sulphate. The material was pooled as indicated. (I) Fraction I. (II) Fraction II. Elution profiles represent typical patterns obtained for samples from each group.
age group. In fraction I, the incorporation of [35S]sulphate an [3H]-glucosamine increased with advance in age, while the reverse was observed in proteoglycan fraction II where the incorporation of both labels significantly decreased with age. Fraction II from the DEAE-Sephacyl column was further examined on Bio-Gel A-50, while fraction I,
25
FRACTION
4 WEEKS
6 WEEKS . . . . . . . . . 8 WEEKS -
0
4
-
- -
(2 WEEKS 18 WEEKS
50
75
101
NUMBER
Fig. 3. Bio-Gel A-50 column chromatography of gingival proteoglycan fraction II from DEAESephacel. Samples of proteoglycans from different age groups were applied separately to a column (0.9 x 120 cm) and eluted with 6 M urea. Fractions of 1.2 ml were collected and monitored for [‘HIglucosamine and [%I-sulphate by scintillation spectrometry. (A) Fraction IIA; (B) fraction IIB. Elution profiles are typical patterns from each age group.
because of the limited amounts, was analysed for glycosaminoglycans directly. Representative patterns on A-50 gel chromatography of the gingival proteoglycans in fraction II are shown in Text Fig. 3. Each age group had two distinct proteoglycan populations. The excluded fraction, fraction IIA, contained the
Gingival proteoglycans
327
L
w
_ (B)
q
4 WEEKS 6 WEEKS
-
l-
TOTAL
HA
GAG
Fig. 4. Distribution of hyaluronic acid (HA) and chondroitinase ABC-digestible glycosaminoglycans (GAG) in proteoglycan fraction 1 (Text Fig. 2) of rat gingiva. Panel A, incorporation of [3H]-glucosamine; panel B, incorporation of [35S]-sulphate. Bars represent the means + SD of values obtained for each age group.
Table 1. Distribution of [“%I-sulphate and [3H]-glucosamine incorporation in gingival proteoglycan fractions I and II from a DEAE-Sephacel column dis/min/g wet weight Age (weeks) 4 6 8 12 18 4 6 8 12 18
Fraction I
Fraction II
[35S]-Sulphate 7800 f 840 194,000 f 18,400 8400 & 860 131,000 + 13,050 12,lOOk 1180 87,700 + 9100 14,200 It 1500 48,300 k 5000 18,600 k 1820 32,000 k 3500 [3H]-Glucosamine 72,000 * 7900 82,000 + 8800 124,000 k 12,000 146,000 + 14,100 156,000 k 15,400
369,000 k 35,000 249,000 + 26,100 167,000 f 16,200 124,000 f 12,300 104,OOOf 11,150
Each value is the mean f SD of triplicate analyses made on each sample.
high-molecular weight (M, > 2 x 106) proteoglycan population, while the included fraction, Fraction IIB, represented the lower-molecular weight (M, < 2 x 106) proteoglycan population. Both populations were present in similar proportions in all age groups. The glycosaminoglycan analyses, made on the proteoglycan fraction I from DEAE-Sephacyl and on the two different molecular-weight proteoglycan populations (IIA and IIB) obtained from fraction II after Bio-Gel A-50 column chromatography, are summarized in Text Figs 4 and 5. The glycosaminoglycans in proteoglycan fraction I showed an agedependent decrease in hyaluronic acid content, and a considerable increase in dermatan sulphate and chondroitin 4- and 6-sulphates. At 4 weeks of age the hyaluronic acid accounted for 46.2% of glycosaminoglycans and dermatan sulphate and chondroitin 4and 6-sulphates for 56.6%; in the l&weeks-old group, the hyaluronic acid amounted to 12.2% of
M. WEINSTEIN et al.
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m 4 WEEKS
2000
a6 m
.c_ 2 h ? .5 E \ 9
D Id 2
WEEKS 6 WEEKS
tDI2 WEEKS
q l8WEEKS
1000
0
5 5000 : 3 0 ; 2500
0
J
TOTAL
DS
c 4-s
c 6-S
HS
I (B)
J TOTAL
DS
c 4-s
C 6-S
HS
Fig. 5. Distribution of [‘HI-glucosamine and [‘5S]-sulphate in glycosaminoglycans of the high molecularweight (fraction IIA) and the low molecular-weight (fraction IIB) proteoglycan populations derived from fraction II after column chromatography on Bio-Gel A-50 (Text Fig. 3). (A) Glycosaminoglycans of Fraction IIA. (B) Glycosaminoglycans of fraction IIB. Bars represent the means + SD of values obtained for each age group. DS, C4-S, C6-S, C6-S and HS denote dermatan sulphate, chondroitin Csulphate, chondroitin 6-sulphate and heparan sulphate, respectively.
glycosaminoglycans, and dermatan sulphate and chondroitin sulphate 4- and 6-sulphates to 86.4% (Text Fig. 4). The data on glycosaminoglycans of the high moleeular-weight proteoglycans excluded from Bio-Gel A-SO, (fraction llA, Text Fig. 3), showed that most of the [35S]-sulphate and [3H]-glucosamine had heen incorporated into dermatan sulphate, followed by
chondroitin 4-sulphate, heparan sulphate, and chondroitin 6-sulphate. None of the age groups showed
the presence of keratan sulphate, and the pattern for each glycosaminoglycan detected remained essentially constant with age. In the 4-week age group, the dermatan sulphate accounted for 60.7% of glycosaminoglycans, heparan sulphate for 10.2%, chondroitin 4-sulphate for 20.1% and chondroitin
Gingival proteoglycans
329
6-sulphate for 3.0%; in the 18-week age group 56.1% of glycosaminoglycans were represented by dermatan sulphate, 20.3% by chondroitin 4sulphate, 10.8% by heparan sulphate, and 3.1% by chondroitin 6-sulphate. Even higher proportions of dermatan sulphate were found among the glycosaminoglycans of the proteoglycan fraction IIB that was included on Bio-Gel A-SO (Text Fig. 3). The glycosaminoglycans of this fraction contained 73-76% of dermatan sulphate, 8-9% chondroitin 4-sulphate, 4.2-4.7% chondroitin 6-sulphate, and 3.54.4% heparan sulphate. However, in comparison to fraction IIA, the glycosaminoglycans of fraction IIB contained about 50% less heparan sulphate (Text Fig. 5).
ageing (Sakamoto et al., 1978). We show that in rat gingival tissue, with increasing age, there is a definite decrease in the synthesis of hyaluronic acid and a pronounced fall in dermatan sulphate, chondroitin 4and 6-sulphates, and heparan sulphate. These data thus lend further support to the notion that the sulphation patterns of glycosaminoglycans and proteoglycans in gingiva may correlate more closely with the stage of growth and development than the currently used indices for the prediction of craniofacial growth.
DISCUSSION
REFERENCES
Our findings clearly show a change in the proteoglycan patterns of rat gingival tissue with age. Although all age groups showed, on DEAESephacel, the presence of undersulphated and sulphated proteoglycan fractions, the undersulphated fraction increased significantly with age, while the sulphated fraction showed a marked decrease. This closely matches the findings of Tian et al. (1986): for the proteoglycans of bone matrix, under similar conditions, they found a small fraction of [3sS]labelled material eluted with 0.28 M NaCl and a major fraction with 0.68 M NaCl; and, as in our study, after gel chromatography, they found two peaks for two different proteoglycan populations. The earlier work on the identification and quantitation of the glycosaminoglycans in gingiva has produced a variety of findings. Only chondroitin 4- and 6-sulphates were identified in digests of human gingiva by Ciancio and Mather (1971). Subsequently, dermatan sulphate, chondroitin 4-sulphate, hyaluronic acid and heparan sulphate were detected in porcine gingiva, but not chondroitin 6-sulphate (Hiramatsu, Abe and Minami, 1978). Other studies with human gingiva have shown variable amounts of dermatan sulphate and chondroitin 4- and 6-sulphate (Bartoid, 1987). We found that the major glycosaminoglycan of rat gingiva is dermatan sulphate, with chondroitin 4- and 6-sulphates, hyaluronic acid, and heparan sulphate also present, but keratan sulphate undetected. These findings are thus in agreement with those Bartold et al. (1981, 1987) and of Dahllof et af., (1986). A progressive decrease in proteoglycan synthesis with age in the embryonic chick has been reported by Robinson and Dorfman (1969) and Kimata et al. (1974). Developmental or age-associated changes in glycosaminoglycans show a decrease in the ratio of chondroitin 4- and 6-sulphates in chick epiphyseal cartilage (Kimata et al., 1974; Robinson and Dorfman, 1979) and an increase in chondroitin 6sulphate in the auricular cartilage of the rabbit (Madsen et al., 1983). For gingival tissue, investigations on material from cows ranging in age from 2 weeks to 1Oyr revealed a rapid decrease in glycosaminoglycans during the first 3 h of development followed further by less evident changes with
Bartold P. (1987) Proteoglycans of the periodontium: structure, role and function. J. periodont. Res. 22, 431-444. Bartold P., Wiebkin 0. and Thonard J. (1981) Glycosaminoglycans of human gingival epithelium and connective tissue. Connecl. Tiss. 9, 99-106. Bitter T. and Muir H. (1962) Determination of uranic acid using a carbozole reagent. Analyt. Biochem. 4, 330-334. Brandt K. D., Palonski M. J. and Perricone E. (1976) Aggregation of cartilage proteoglycans. II. Evidence for the presence of a hyaluronate-binding region on proteoglycans from osteoarthritic cartilage. Arfhrifis Rhem. 19, 1308-1314. Ciancio S. and Mather M. (1971) Acid mucopolysaccharides in gingivitis and periodontitis. J. periodont. Res. 6, 188-193. Dahllof G., Modeer T., Reinholt F., Wikstrom B. and Hjerpe A. (1986) Proteoglycans and glycosaminoglycans in phenytoin-induced gingival overgrowth. J. periodont. Res. 21, 13-21. Greulich W. and Pyle S. (1959) Radiographic Atlas of Skeletal Development of the Hand and Wrist, 2nd edn. Stamford University Press. Stanford, CA. Hiramatsu M.. Abe I. and Minami N. (1978) Acid mucopolysaccharides in porcine gingiva. J. ieriohont. Res. 13, 224-227. Houston N., Miller J. and Tanner J. (1979) Prediction of the timing of adolescent growth spurt from ossification events in hand-wrist films. >r. J. &ho. 6, 145-152. Kimata K.. Okavama M.. Oohira A. and Suzuki S. (1974) Heterogeneity of protedchondroitin sulfates produced by chondrocytes at different stages of cytodifferentiation. J. biol. Chem. 249, 16461653. Madsen K., Maskalewski S., Von Der Mark K. and Frieberg U. (1983) Synthesis of proteoglycans, collagen and elastin by culture of rabbit auricular chondrocytesRelation to age of the donor. Devl Biol. 96, 63-73. Nakano T. and Scott P. G. (1989) A quantitative chemical study of glycosaminoglycans in the articular disk of the bovine temporomandibular joint. Archs oral Biol. 34, 749-757.
Acknowledgement-This work was supported by Grant DE05666-I3 from the National Institute of Dental Research, National Institutes of Health.
Robinson H. and Dorfman A. (1969) The sulfation of chondroitin sulfate in embryonic chick cartilage epipyses. J. biol. Chem. 244, 348-352. Saito H., Yamagata T. and Suzuki S. (1968) Enzymatic methods for the determination of small quantities of isomeric chondroitin sulfates. J. Biol. Chem. 243, 15361542. Sakamoto N., Okamoto H. and Okuda K. (1978) Qualitative and quantitative analysis of bovine gingival glycosaminoglycans. Archs oral Biol. 23, 983-987.
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Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J. and Klenk D. C. (1985) Measurement of protein using bicinchoninic acid. Analyf. Biochem. 150, 7685. Tian M., Yanagishita M., Hascall V. and Reddi A. (1986) Biosynthesis and fate of proteoglycans in cartilage
and bone during development and mineralization. Arch. Biochem. Biophys. 241, 221-232. Wasserman L., Ber A. and Allalouf D. (1977) Use of thin-layer chromatography in the separation of disaccharides resulting from digestion of chondroitin sulphates with chondroitinases. J. Chromatog. 36, 342-347.
