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Azerbaycan Saytlari

 »  Home  »  Endodontic Articles 13  »  Location, arrangement and possible function of interodontoblastic collagen fibres in association with calcium hydroxide-induced hard tissue bridges
Location, arrangement and possible function of interodontoblastic collagen fibres in association with calcium hydroxide-induced hard tissue bridges
Introduction - Materials and methods.



Y. Kitasako, S. Shibata, C. F. Cox & J. Tagami
Cariology and Operative Dentistry, Department of Restorative Sciences and Maxilofacial Anatomy, Department of Maxillofacial/ Neck Reconstruction Maxillofacial Biology, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan.
Departments of Restorative Dentistry and Biomaterials, University of Alabama, Birmingham, AL, USA.


Introduction.
A biological and/or mechanically tight bacteriometic seal is essential for an orderly progression of cell proliferation, contact recognition against the agent interface and eventual bridge formation between the restoration interface and the capping agent (Cox et al. 1982, Tziafas et al.1992b, Kitasako et al. 2000a). Fitzgerald (1979) followed cellular reorganization in mechanically exposed and directly capped monkey pulps. He confirmed that dentine bridge formation occurred when the capping agent was placed indirect contact with the pulp, the cells following a pattern of cell proliferation, stratification and polarization to form new odontoblastoid cells and matrix formation, without being preceded by a zone of necrosis. Routine use of Ca(OH)2 and certain other ‘bacteriometic’ agents permits cell organization and differentiation of new odontoblastoid cell formation directly adjacent to the agent interface (Cox et al.1982,1987).
In von Korff’s original paper (1906), an argyrophilic interodontoblastic fibre (von Korff fibre) arose from the dental pulp, passing between the odontoblasts and fanning out to form the fibrous matrix of the dentine. In general, von Korff fibres may help to guide the odontoblasts in their pulp ward migration during dentinogenesis and to bind the soft tissue pulp and odontoblasts to the dentine. Lester & Boyde (1968) have cited evidence that von Korff fibres are present during secondary dentinogenesis. The micrographs of Silva & Kalis (1972) and Reith (1968) also suggested that the fibres might be present in early circumpulpal dentine formation, whilst Furseth (1971) reported that they might be present later in the roots of human teeth.
In his original paper, von Korff (1906) described argyrophilic von Korff fibres as radiating between the primary odontoblasts and fanning out near their apical neck to take part in the formation of the collagen matrix of the developing intertubular substrate. Silva & Kalis (1972) suggested that von Korff fibres were present in early circumpulpal dentine formation. Hayward & Webb (1984) demonstrated collagen bundles coursing from the pulp into the predentine substrate suggesting that von Korff fibres are present in all stages of dentinogenesis.
Recent transmission electron microscopy and scanning electron microscopic studies show the existence of interodontoblastic collagen fibres in human dentine (Sogaard-Pedersen et al.1990), in the root of cat dentine (Bishop et al.1991) and in mechanically exposed monkey dental pulp (Kitasako et al. 2000a). A parallel study by Salmon et al. (1991) demonstrated bundles of fibrils that course between odontoblasts through the predentine to become incorporated into the mineralized dentine. Following experimental pulpotomy capping with a Ca(OH)2 slurry in dogs, Higashi & Okamoto (1996)postulated that these interodontoblastic fibrils provide support to the cells before their formation of odontoblasts.
However, some controversy remains over these fibres as Ten Cate (1998) suggests the term von Korff fibre to be incorrect. Consequently, the current mechanical or biological nature of von Korff fibres or von Korff-like fibres (VKF) remains unclear; however, due to their frequent occurrence, they appear an important source of collagen for dentine substrate. The aim of this study was to assess the location, arrangement and possible function of interodontoblastic collagen fibres in association with calciumhydroxide-induced hard tissue bridges by using light and transmission electron microscopy techniques and immunohistochemical staining localization.

Materials and methods.

Animals.
Two monkeys (Macaca fuscata), both aged 7 years and showing no evidence of dental or periodontal disease, were housed in facilities approved by the Tokyo Medical and Dental University. The animal use protocol form was reviewed and approved by the Screening Committee for Animal Research of the Tokyo Medical and Dental University, prior to the study. Because five different observation periods (3, 14, 21, 30 and 90 days) were used, two primates were divided into two groups: one was tested at 3, 21and 90 days and the other one was tested at 3, 14 and 30 days after operation, respectively (Table 1).

Table 1. Numbers of tested teeth for each animal.

Numbers of tested teeth for each animal

Experimental procedures.
Each monkey was subjected to general anaesthesia by an intramuscular injection of 20 mg kg_1 ketamine (Ketaral; Sankyo Co., Tokyo, Japan), followed by an intravenous injection of 10 mg kg_1 pentobarbital sodium (Nembutal; Sodium Solution, Abbott Laboratories, Abbott Park, IL, USA). Class V cavities were prepared in descending order of the length of observation periods (10 teeth per time period) with a high-speed tapered diamond bur (number 170; GC Co., Tokyo, Japan) under water-spray coolant. Each pulp was intentionally exposed on the cavity floor with a round carbide bur (number1; Shofu Inc., Kyoto, Japan), 0.8 mm in diameter. Infiltration anaesthesia was performed in the adjacent gingiva of the exposed pulps with lidocaine hydrochloride containing 1: 80000 adrenaline (Xylocaine; Fujisawa Co., Osaka, Japan) to control haemorrhage and exudate from the exposure site. Each exposed pulp was immediately capped with a hard-set Ca(OH)2 (Dycal; L. D. Caulk Co., Milford, DE, USA). The base and catalyst were mixed and applied directly onto the exposed pulps. Effort was made to keep all dentine and cavity walls free of the Ca(OH)2 agent. After the Ca(OH)2 had set, each cavity was then sealed with an adhesive resin system (Clearl Liner Bond II V; Kuraray Co. Ltd, Osaka, Japan). Each cavity was first conditioned with LB Primer for 30 s, air-dried and then coated with LB Bond and photo-cured for 20 s. Protect Liner F, a low-viscosity resin composite, was then applied to the cavity floor, covering the Ca(OH)2 agent and photo-cured for 20 s. All cavities were restored to the cavo surface margin with a hybrid resin composite (Clearl AP-X; Kuraray Co. Ltd, Osaka, Japan) and photo-cured for 30 s.

Tissue preparation.
At the end of the experimental observation periods, both monkeys were killed with an intravenous injection of 250 mg kg_1 thiopental sodium (Ravonal, Tanabe Pharmaceutical Co., Osaka, Japan). The teeth were immediately removed by surgical extraction and immersed in 4% paraformaldehyde (PA) solution (0.1m phosphate buffer, pH 7.4) for light microscopy or 5%glutaraldehyde (GA)_4% paraformaldehyde (PA) solution (0.1m phosphate buffer, pH 7.4) for transmission electron microscopy at room temperature for 2 days. Twenty-five PA-fixed specimens were demineralized in10%EDTA (Ethylenediamine- N,N,N0,N0-tetraacetic acid) at 4 8C for 2 weeks and embedded in parafin. Twenty GA-PA-fixed specimens were demineralized with EDTA, if required, and post-fixed in1% osmium tetroxide (0.1m phosphate buffer, pH 7.4, 4 8C) for 3 h, rinsed and embedded in Rigolac resin (Oken,Tokyo, Japan).

Silver staining and immunohistochemistry.
Serial parafin sections (4 mm) were cut and the tissues silver impregnated by Gomori’s reticulin method (Gomori1937) to identify collagen fibres. Immunohistochemistry was performed as previously described (Shibata et al. 1997). Briey, after deparafinization, the sections were immersed in PBS and digested with testicular hyaluronidase (25 mg mL_1; Sigma Chemicals) in PBS for 30 min at 37 8C. After several washings in PBS, the sections were immersed in methanol containing 1% hydrogen peroxide and 1% bovine serum albumin to block endogenous peroxidase activity and nonspecific reactions. Sections were then incubated with commercial rabbit polyclonal antibodies (LSL, Tokyo, Japan) against collagen type I (antibovine type I collagen), III (antiratmultiple antigen peptide), or fibronectin (antirat multiple antigen peptide), diluted 1:1000 with PBS. The streptavidin-biotin method was applied to the sections using HISTOFINE SAB kits (Nichirei, Tokyo, Japan). The sections were treated with 3-amino-9-ethylcarbazole to reveal any reaction. Negative control sections for immunohistochemistry were incubated with normal rabbit IgG instead of the primary antibodies. Sections were observed after counterstaining with haematoxylin.

Transmission electron microscopy.
One micrometer mm resin sections were cut and stained with toluidine blue for light microscopy to confirm the orientation. Ultrathin sections were then cut and contrasted with phosphotungstic acid, uranyl acetate and lead citrate and examined in a Hitachi H-800 transmission electron microscope.