Microfibrillar components in dental pulp: Presence of both type VI collagen- and fibrillin-containing microfibrils

Arch oral Biol. Vol. 37, No. 12. pp. 1079-1084, 1992 Printed in Great Britain. All rights reserved

Copyright c

0003s9969/92 $5.00 + 0.00 1992 Pergamon Press Ltd

MICROFIBRILLAR COMPONENTS IN DENTAL PULP: PRESENCE OF BOTH TYPE VI COLLAGEN- AND FIBRILLIN-CONTAINING MICROFIBRILS C. Department

A.

SHUTTLEWORTH.

of Biochemistry

LINDA BERRY

and

CAY M. KIELTY

and Molecular

Biology. Manchester University. Stopford Building, Oxford Road. Manchester Ml3 9PT. U.K.

(Receired

7 April

1992; accepted

16 June

1992)

Summary-Microfibrillar

elements were isolated from developing and formed bovine dental pulp by a procedure involving bacterial collagenase tissue digestion and chromatography on Sepharose CL-2B. Two microfibrillar assemblies could be demonstrated. Type VI collagen microfibrils with a characteristic periodicity of about 100 nm appeared as long, thin. flexible filaments. In a number of cases these structures aggregated by lateral association. Microfibrils of IO-14 nm dia were identified as containing fibrillin on the basis of their distinctive, periodic. beaded morphology. In addition to long. single strands there were instances of chains coalescing to give amorphous aggregates. No differences in the type of microfibrillar assemblies were evident between developing and formed pulp. although fibrillin-containing microfibrils were more abundant in formed pulp. Key words: dental pulp. mlcrofibrils. type VI collagen. fibtillin

INTRODUCTION

Dental pulp is a specialized connective tissue that functions in the production and maintenance of dentine. In general, pulp is considered to be low in collagen. The composition of the matrix changes with age, the components of the ground substance decreasing in favour of an increased collagen content (Zerlotti, 1964). Pulp fibres have been classified histologicall! as reticular fibres, including von Korff fibres. collagen fibres and elastic fibres. the last always being associated with the larger blood vessels but never the capillaries (Baume, 1980). This classification relies on staining and morphological characteristics and does not positively identify the nature of the components. Cross-banded collagen fibres can be clearly seen. and both type I and III collagen account for the bulk of the tissue collagen (Shuttleworth. Ward and Hirschman, 1978; Shuttleworth. Berry and Wilson. 1980). The nature of other fibrillar material remains contentious, but the staining and morphological characteristics of these assemblies suggest that they may be related to microfibrillar components. which are ubiquitous throughout connective tissue matrices. In recent years both collagenous and non-collagenous microfibrillar networks have been characterized. and both type VI collagen and fibrillin shown to form distinct macromolecular assemblies (Kielty et al.. 1991). Type VI collagen is essentially a glycoprotein with a short, triple-helical core and large. N- and C-terminal globular domains. Collagen VI monomers are assembled into tetramers before their secretion from the cell and these are assembled end-on-end in the extracellular space into periodic microfibrils (Engvall, Hessle and Klier, 1986; Colombatti tar a(.. 1987).

Fibrillin has recently been identified as a single-chain glycoprotein which assembles into periodic microfibrils with a wide distribution in both elastic and non-elastic tissues (Gibson, Kumaratilake and Cleary. 1989; Sakai, Keene and Engvall. 1986; Maddox er al., 1989; Keene et al.. 1991: Sakai et al., 1991). A structure based on the overlap of units, each consisting of a bead with fine filaments extending from them. has been proposed (Wright and Mayne, 1988). We have recently described a method for the selective isolation of intact microfibrillar arrays of both type VI collagen and fibrillin from bovine skin (Kielty et al.. 1991). In an attempt to characterize the microfibrillar elements in pulp, we have now applied this technique to erupted and unerupted bovine molars. and have examined the microfibrils isolated from the dental pulp. MATERIALS AND METHODS

Fetal calves and jaw bones from 2-yr-old steers were obtained from the local abattoir within I h of death. Jaw bones were split and the developing tooth pulp removed. or the teeth extracted and pulp taken from the cracked tooth. Microfibrillar proteins from dental pulp were prepared by the method of Kielty et al. (1991). In brief. pulp samples (approx. I g wet weight) were washed in phosphate-buffered saline and homogenized in 5 ml 0.05 M tris-HCI, pH 7.4, containing 0.4 M NaCI, 0.01 M CaCl,, 2 mM phenylmethanesulphonyl fluoride and 10 mM N-ethylmaleimide. Bacterial collagenase (Sigma type IA), final concentration 0.2 mg/ml, was added and the tissue digested at room temperature for 4 h with gentle stirring. The reaction was terminated by the addition of 10 mM

1079

C.

A.

Sepharose

%~I.JTTLEWORTH

CL-

28

er al.

chromotograohy

2000

1500

$ Gi l-u

1000

IYi

Froctlon

NO

Fig. I. Elution profile from Sepharose CL-2B column (1.5 x 100 cm) eked containing

0.4 M NaC1. Samples were chromatographed Developing pulp from 66cm fetus; 0.

EDTA and the solution clarified by centrifugation (10,OOOg for 1 h). Two mg DNase (Boehringer Mannheim-bovine pancreas) and 250 units leech hyaluronidase (EC 3.2 1.36; Sigma Chemical Co., St Louis, MO. U.S.A.) were added, the supernatant made 10mM in MgCl,. digested overnight at 4’C and applied directly to a Sepharose CL-2B column. In some cases no leech hyaluronidase was added before chromatography. Gel-filtration chromatograph?,

Samples were chromatographed directly without concentration under non-reducing. non-denaturing conditions on a column (I .5 x 100 cm) of Sepharose CL-2B equilibrated with 0.05 M-tri-HCl. pH 7.4. containing 0.4 M NaCl, at a flow rate of 12 ml/h. Column effluent was monitored at 280 nm. 3.2ml fractions were collected and appropriate fractions pooled. Antibody production

Polyclonal antibodies to intact microfibrillar proteins were raised in rabbits. Antiserum to intact type VI collagen microfibrils recognized the z l/r2 chains of type VI collagen (Kielty et al.. 1991). and did not cross-react with fibrillin. An antibody to fibrillin was generated by injecting a fibrillin-rich microfibrillar preparation from skin into rabbits. It has been proposed that fibrillin-containing microfibrils contain a number of proteins (Gibson et al.. 1989). but the antisera used recognized fibrillin and type VI collagen chains on Western blotting. and a single component (M, 300 K) could be immunoprecipitated from Plate Fig. 2. Electron micrographs adult pulp. Extenstve arrays

with 50 mM WIS-HC1, pH 7.4, at 12 ml/h and 3.2 ml fracttons collected. 0. dental pulp from adult steer.

human smooth-muscle cell cultures. Immunogold localization with this antibody clearly showed that it recognized an epitope on the native, intact. fibrillincontaining microfibrils and did not appear to react with type VI collagen microfibrils. Rotary shadowing electron microscop?

The content of high M, extracellular matrix macromolecules was monitored by examination of samples by rotary shadowing. Samples were diluted directly into 0.2 M ammonium acetate, pH 6.0. to a final concentration of approx. 100 pgjml and analysed as described by Kielty ef al. (1991). Immunogold localization

Protein-antibody-gold complexes were prepared from the Sepharose CL-ZB- excluded peak using a modification of the method of Sheehan er al. (1987). In brief. a 250 p I sample was mixed with 250 p 1 of antiserum (diluted 1: 500 in 5 mM magnesium acetate, pH 7.0;0.1% Tween). After 1 h, excess antibody was removed by chromatography on Sepharose CL2B (150 x 10 mm) equilibrated in 20 mM magnesium acetate and run at 0.5 ml/min. The protein-antibody complex was collected from the void-volume peak, diluted with 10ml 20mM magnesium acetate and prepared for electron microscopy as described above. Before rotary shadowing, grids were washed in 50 ~1 droplets of protein A-gold (diluted l/l0 in 5 mM magnesium acetate/l % Tween), (gold particles 5 nm; Janssen Scientific) washed 4 x with 50 ~1 droplets of 5 mM magnesium acetate/O.]% Tween, once with 5 mM magnesium acetate and finally in 95% ethanol. I

after rotary shadowing of intact microfibrils of type VI collagen isolated from of type VI collagen are present (A, B). Immunogold localization of antibody to type VI collagen (C). Bar 100 nm.

Dental pulp microfibrils

.<‘. :

, .

. _

r

-

Plate I

1083

Dental pulp microfibrils After drying in air, grids were rotary shadowed with platinum/tungsten at an angle of 8” and examined on a Jeol 1200EX electron microscope at 120 kV. Type VI collagen samples prepared in this way appeared more compact after rotary shadowing.

RESULTS

Digestion of either developing or formed pulp with bacterial collagenase effectively solubilized all of the tissue into 0.4 M NaCl buffer. Fractionation of these digests directly on Sepharose CL-2B clearly showed that molecular assemblies in excess of 2 x lo6 could be isolated by this means (Text Fig. 1). In all cases the majority of u.v.-absorbing material eluted at or near the column volume, as expected for plasma proteins and collagenase-susceptible material. Examination of the high M, material isolated from the Sepharose column by rotary-shadowing electron microscopy showed that both type VI collagen- and fibrillin-containing microfibrils were present (Plate Figs 2 and 3). Type VI collagen, the most abundant high M, component in the excluded peak was present in the form of long, thin microfibrils with a periodicity and internal dimensions consistent with a construction of tetrameric units arranged end to end [Plate Figs 2(A) and 2(B)]. Immunogold localization with an antibody to type VI collagen showed periodic epitope recognition and served as additional confirmation of the identity of these microfibrils [Plate Fig. 2(B)]. Similar assemblies were seen in samples prepared from developing pulp tissue (data not shown). Fibrillin was present and easily detectable: microfibrils composed of repeating, compact, electron-dense globes with a number of long, thin filaments extending from them had the morphological characteristics of elements shown to contain fibrillin [Plate Fig. 3(A)]. In addition to single-chain assemblies a number of amorphous aggregates were evident. indicating that the single chains could coalesce [Plate Fig. 3(B)]. On examination of a number of grids from developing and formed pulp it became apparent that there was an increase in the frequency of fibrillin-containing microfibrils. Immunogold localization with the type VI collagen antibody failed to stain any of the fibrillin-containing microfibrils. whereas epitopes were clearly present on the surface of the periodic. compact-beaded domains when the fibrillin antiserum was tested [Plate Fig. 3(C)]. Similar assemblies were seen in samples prepared from developing pulp tissue (data not shown). DISCUSSION

Microfibrils are ubiquitous components of connective tissue and can be released from tissues as intact,

high M, assemblies by removal of the fibrillar collagen network with bacterial collagenase. We clearly show here that both type VI collagen- and fibrillin-containing microfibrils are present in pulp. Type VI collagen exists as extremely long polymers. which are highly flexible and capable of lateral aggregation. Higher-order structural arrays have been described in skin and tendon (Bruns et al., 1986; Keene, Engvall and Glanville, 1988). Collagen VI microfibrils were similar in developing and formed pulp, although lateral aggregates appeared to be more common in formed pulp preparations. We have previously shown that type VI collagen microfibrils in skin associate with hyaluronan (Kielty et al., 1992). Hyaluronidase treatment of pulp samples eliminated filaments of hyaluronic acid associated with the type VI collagen microfibrils (data not shown). The ubiquitous distribution of type VI collagen and its ultrastructural localization around collagen fibrils and at the surface of cells implicate this molecule in matrix formation and remodelling. In preliminary studies we find that type VI collagen appears to be enriched in the subodontoblastic layer and it is interesting to speculate that these microfibrils may be the principal component of von Korff fibres. Fibrillin-containing

microfibrils

were detected

in

pulp samples at all stages of development, and appeared to increase in amount during pulp formation. A number of protein components have been implicated in the structure of these microfibrils (Gibson et al., 1989), but monoclonal antibodies to fibrillin bind in a periodic manner (52 nm) to them, serving to confirm that fibrillin is a structural component (Maddox et al., 1989; Sakai ef al., 1986). Fibrillin has been associated with the deposition of elastin, although histological stains suggest that little elastin is present in dental pulp. However, it is becoming increasingly apparent that fibrillin is present in non-elastic tissues, which implies an additional role(s) for this microfibrillar component unrelated to elastic deposition. Considerable difficulties are encountered in trying to characterize fibrillar elements in connective tissue by histological staining properties. It is clear from a number of studies that collagens I and III are the main fibrillar elements present in dental pulp matrix (Shuttleworth, 1990). Our work clearly identifies two microfibrillar components that can be readily isolated from dental pulp. Both type VI collagen- and fibrillinmicrofibrils are widely distributed containing throughout extracellular matrices, although the potential biological roles of both of these elements remains to be established. Their presence in dental pulp suggests that these structures are involved in both development and maintenance of the pulp tissue. Further work will be required to establish the organization and function of these important elements in dental pulp.

Plate 2 Fig. 3. Electron micrographs after rotary shadowing of intact microfibrils of fibrillin isolated from adult pulp. Long, flexible chains of fibrillin can be seen (A) which often associate to form amorphous aggregates (B). Immunogold localization of antibody to fibrillin (C). Bar 1OOnm.

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