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 »  Home  »  Endodontic Articles 10  »  Calcium sulphate as a bone substitute for various osseous defects in conjunction with apicectomy
Calcium sulphate as a bone substitute for various osseous defects in conjunction with apicectomy
Introduction - Materials and methods.

Y. Murashima, G. Yoshikawa, R.Wadachi, N. Sawada & H. Suda
Department of Restorative Sciences, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan.

Osseous defects associated with infected tooth roots vary in their type and size: the most common are endodontic- periodontal osseous defects, large osseous defects, or ‘throughand through’osseous defects. Ideally following treatment, these defects should become completely filled with new bone; however, they often heal with fibrous scar tissue.
The principle of guided bone regeneration (GBR) has been applied to osseous defects in conjunction with apicectomy (Dahlin et al.1990, Pecora et al.1995,Yoshikawa et al. 2002). Dahlin et al. (1990) reported that GBR using expanded polytetrafluoroethylene (e-PTFE) membrane had the ability to predictably achieve bone regeneration. Yoshikawa et al. (2002) reported that e-PTFE membrane was most effective on bone regeneration in osseous defects in conjunction with apicectomy when compared to two kinds of resorbable membranes and a control. The membranes, however, are not easy to apply on the complicated osseous defects, and the e-PTFE membranes must be removed with a second surgical procedure because they are not resorbable.
Autogenous bone grafting has been shown to be effective for treating bone loss in orthopaedic surgery (Rodriguez et al. 1995), but this technique is limited by the finite amount of bone available, and osteotomy for obtaining the autogenous bone causes patient discomfort. Therefore, bone substitutes may be more effective for encouraging bone regeneration. The ideal bone substitute should be
  1. osteogenic,
  2. biocompatible,
  3. bioresorbable,
  4. able to provide structural support,
  5. easy to use clinically,
  6. cost-effective (Tay et al. 1999), and
  7. able to be sterilized without any changes in its properties.
Calcium sulphate has been used as a bone substitute in orthopaedics and oral surgery for many years (Dreesmann1892,Peltier1961,Bahn 1966, Shaffer & App 1971, Conner 1996, Andreana 1998). This material is resorbable (Peltier 1961, Bell 1964), biocompatible (Mesimeris et al. 1995, Sottosanti 1995), and useful as a binder or carrier of other materials (Yamazaki et al. 1988, Najjar et al. 1991, Pepelassi et al. 1991, Sottosanti 1992 a, b, Dibattista et al.1995, Sottosanti 1995, Anson1996, Bai et al.1996, Benoit et al.1997, Sottosanti 1997, Kim et al. 1998, MacNeill et al. 1999, Rosen & Reynolds 1999). It has been suggested that calcium sulphate could be substituted for e-PTFE membrane in bone regeneration after apicectomy (Yoshikawa et al. 2002). Calcium sulphate is applied easily without reflecting the palatal or lingual flap especially in ‘through and through’ osseous defects. Pecora et al. (2002) reported that calcium sulphate improved the clinical outcome in the case of ‘through and through’osseous defects. As healing was evaluated radiographically in that study, the nature of the tissues that had formed within the defects was unknown. Despite these studies, the role of calciumsulphate in bone regeneration has not been clarified. The purpose of this study was to investigate the effect of calcium sulphate on various osseous defects when used in conjunction with apicectomy.

Materials and methods.
Eleven beagle dogs were used in this study. Permission for this study was given by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University. The dogs were sedated by an intramuscular injection of ketamine hydrochloride (Ketalar1, 17 mg kg_1, Sankyo Co Ltd, Tokyo, Japan) and deeply anaesthetized by an intravenous injection of sodium thiopental (Ravonal1, 25 mg kg_1,Tanabe Seiyaku Co Ltd, Osaka, Japan). A deep level of anaesthesia was maintained by supplementary doses of the same drug when necessary. Local anaesthesia, 2% lidocaine with 1: 80 000 epinephrine solution (Xylocaine1, Astra Japan Ltd, Osaka, Japan), was injected into the site of surgery to control haemorrhage.
Once the dogs were anaesthetized, the root canals of the mandibular third and fourth premolars on both sides were instrumented with K-files (sizes 10-50) and Gates-Glidden burs (sizes 2-4), whilst being irrigated with 6% sodium hypochlorite and 3% hydrogen peroxide. After the root canals were dried with paper points, they were obturated with gutta-percha and root-canal sealer (Canals-n1, ShowaYakuhin Kako Co Ltd, Tokyo, Japan) by the lateral condensation method. The access cavities were sealed with glass-ionomer cement (GCFuji Ionomer1 type II, GC Corporation, Tokyo, Japan).
Full mucoperiosteal flaps were then reflected bilaterally from the second premolar to the first molar to expose bone in the apical area of the teeth. Three types of osseous defects were prepared on both sides of the mandible using a trephine bur (4.8 mm in diameter) under water cooling as follows:
  • type1, osseous defect communicating with the periodontal pocket on the mesial root of the third premolar;
  • type 2, large osseous defect including the distal root of the third premolar and the mesial root of the fourth premolar;
  • type 3, ‘throughand through’ osseous defect on the distal root of the fourth premolar (Fig.1).

Figure 1. Diagram of the osseous defects. type1, osseous defect communicating with the periodontal pocket; type2, large osseous defect; type3,'throughand through'osseous defect.

Diagram of the osseous defects

After the roots of the third and fourth premolars were resected using a fissure bur underwater cooling, the retrograde cavities were prepared using an ultrasonic tip (Osada Electric Co Ltd, Tokyo, Japan). Following haemostasis of the osseous defects with 0.1% epinephrine pellets, the retrograde cavities were dried with air and filled with SuperEBA1 (Harry J. Bosworth, Skokie, IL, USA). The osseous defects on the experimental side, which was decided randomly for each dog, were filled with medical grade calcium sulphate (Class Implant, Rome, Italy), and those on the opposite side were left unfilled and acted as controls. The flaps were then repositioned and sutured with 4-0 nylon sutures.
The dogs were injected with pulse labels of tetracycline (20 mg kg_1, Sigma, St.Louis, MO, USA) and calcein (8 mg kg_1, Fluka, Buchs, Switzerland) as hard tissue marking agents at14 and 2 days before sacrifice, respectively. The dogs were sacrificed by an overdose of sodium pentobarbital at 8 and 16 weeks postoperatively. Mandibular blocks including the third and fourth premolars were removed, trimmed and fixed immediately in 8%formalin and 2.5% glutaraldehyde solution for 7 days at 4 8C. The specimens were washed and dehydrated in a graded series of ethanol and embedded in polyester resin (Rigolac1, Nisshin EM, Tokyo, Japan). In the type 1 and 3 defects, undemineralized semi-serial sections (60 mm in thickness) in a longitudinal direction to the root were obtained. In the type 2 defects, sections in horizontal direction through the largest diameter of the defect were obtained. The sections were stained by toluidine blue stain or Villanueva’s bone stain, and evaluated histologically and morphometrically under a light microscope and a fluorescence microscope.

Histological evaluations.
Tissue sections were evaluated histologically with respect to:
  1. the formation of the bone on the buccal side of the root in type 1 defect;
  2. inflammation in the osseous defect; and
  3. bone regeneration within the osseous defect.
Morphometrical evaluations.
Images of all specimens were captured by means of computer software (Adobe Photoshop1, Adobe Systems Inc., San Jose, CA, USA)which dealt with the microscopic pictures, and the morphometrical measurements were performed using an image analysis system (KS4001, Carl Zeiss Co Ltd, Gottingen, Germany).The images were numbered randomly to blind the experimental side, and another examiner performed all the morphometrical evaluations to avoid measurement error.
The measured parameters were defined as follows. Reference points (a-d in Fig. 2 (A), and a-h in Fig. 2(B)), which were represented by the outer and inner edges of the original cortical bone, clearly created the measuring area representing tissue volume (TV). Bone volume/ tissue volume (BV/TV) was defined as the percentage of the total bone within the measured area (Yoshikawa et al. 2002). In type 3 defects, BV/TV was calculated as a total of both the buccal and lingual areas. Mineral apposition rate (MAR) (mm day_1) was defined as the mean distance between the tetracycline line and the calcein line divided by12 (days). The mean distance was calculated by taking the average of the five distances which were measured randomly on every image.
For statistical analysis, the two-way factorial anova was performed (Stat View V, Abacus, Berkeley, CA, USA).

Figure 2. Diagram of morphometrical evaluation (BV/TV). BV/TV represented the rate of regenerated bone (area of dots) within the cortical osseous defect (square marked by solid line).

Diagram of morphometrical evaluation