Derivative in the Treatment of Intrabony Periodontal Defects in Dogs

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Journal International Journal of Periodontics & Restorative Dentistry, 27(5): 471‑479

URL http://hdl.handle.net/10130/421

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Regenerative periodontal procedures are used to restore lost support for the dentition around diseased root surfaces. Various types of graft materials are used to treat intrabony defects;

autografts or allografts are common.^1,2 Bone grafting materials supply a scaffold for host cells along with factors that stimulate regeneration through bone conduction.^3,4Various types of materials have recently satisfied these criteria in human models: intraoral autogenous bone,⁵ demineralized freeze-dried bone allograft (DFDBA),^1,6 human recombinant platelet-derived growth factor (rhPDGF) plus ␤-trical- cium phosphate,⁷ DFDBA plus rhPDGF,^8,9 bovine-derived porous bone xenograft (BDX),¹⁰and enamel matrix derivative (EMD).^11,12

BDX is derived from cancellous bovine bone, and all organic compo- nents and pathogens are removed by chemical extraction.¹³ BDX acts as a scaffold to support the growth of new tissue¹⁴and is subsequently replaced by the host tissue. Successful treatment of periodontal intrabony defects with bone grafting material depends on the size of the defect, as the aim of such procedures is to stimulate regenera-

Combination of Bovine-Derived Xenografts and Enamel Matrix

Derivative in the Treatment of Intrabony Periodontal Defects in Dogs

Shigeki Yamamoto, DDS*

Hiroyuki Masuda, DDS, PhD**

Yoshihiro Shibukawa, DDS, PhD***

Satoru Yamada, DDS, PhD****

The aim of this study was to investigate the effect of a combination of enamel matrix derivative (EMD) and bovine-derived xenograft (BDX). Intrabony defects were created in dogs and treated with BDX plus EMD, with BDX alone, or with neither (control group). Control group defects were characterized by a long junctional epithelium and little bone formation. The BDX+EMD sites showed a statistically significant increase (P < .05) in new bone and cementum formation compared with the BDX-only sites. These findings suggest that the use of BDX with EMD is effective in enhancing new bone and cementum formation and that this combination is effective in the treatment of intrabony defects. (Int J Periodontics Restorative Dent 2007;27:471–479.)

*Graduate Student, Department of Periodontics, Tokyo Dental College, Tokyo, Japan.

**Instructor, Department of Periodontics, Tokyo Dental College, Tokyo, Japan.

***Associate Professor, Department of Periodontics, Tokyo Dental College, Tokyo, Japan.

****Professor and Chairman, Department of Periodontics, Tokyo Dental College, Tokyo, Japan.

Correspondence to: Dr Shigeki Yamamoto, Department of Periodontics, Tokyo Dental College, 1-2-2 Masago, Mihama-ku, Chiba-shi 261-8502, Japan; fax: +81-43-270-3955;

e-mail: [email protected].

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tion through bone conduction.¹⁵ Therefore, application of these procedures is limited to cases of two- or three-wall bony defects; bone grafting therapy is not effective in promoting the closure of advanced intrabony defects.

The induction process necessitates grafting material and growth factors to stimulate host cells for regeneration of lost structures. The concept of applying grafting materials in periodontal regeneration using both bone induction and conduction is, therefore, an attractive one. Animal experiments¹⁶and clinical studies¹⁷have shown that EMD stimulates the regeneration of periodontal tissues, including acellular cementum, periodontal ligament (PDL), and alveolar bone. However, because of its fluid consistency, EMD has limited space- making potential, and the application of bone grafting materials may help avoid flap collapse. EMD is believed to mobilize fibrous stromal cells into spaces formed by bone particles, which are then gradually filled by the newly formed bone.¹⁸In some clinical studies, treatment of intrabony defects with a combination of BDX and EMD was found to improve the clinical outcome of regeneration.¹⁹However, in another recent clinical study,²⁰this combination

produced no difference in its effect on the healing process when compared to treatment with BDX alone. At present, it is not clear whether treatment of intrabony defects with BDX plus EMD offers an advantage over treatment with BDX alone.

The aim of the present study was, therefore, to determine whether the use of BDX, both alone and in combination with EMD, offered an advantage in terms of periodontal tissue regeneration in intrabony defects as an adjunct to BDX treatment.

Method and materials

Twenty-four beagle dogs were used in this study. The animals were placed under general anesthesia with keta- mine hydrochloride at a dosage of 10 mg/kg. Experimental surgery involved elevating buccal and lingual mucoperi- osteal flaps to surgically create four

“box-type” two-wall intrabony defects in each dog (5 ⫻ 5 ⫻ 5 mm) (Fig 1).

Twelve weeks after creation of the defects, the flaps were raised, granulation tissue was removed, and the root surfaces facing the defects were scaled and planed. Using a small round bur,

reference notches indicating the bot- tom of the defect were prepared on the root surfaces. The intrabony defects were then randomly assigned to one of the following three treatments (eight dogs in each group): BDX alone (Bio- Oss, Osteohealth), combined therapy of BDX plus EMD (BDX+EMD; Emdo- gain, Straumann) (Fig 2), and no application of materials as a control group.

In all groups, the root surface was etched for 2 minutes with 24%

ethylenediaminetetraacetic acid (PrefGel, Straumann). At sites that received the combined treatment, EMD was applied to the root surfaces and the defects. After the application of EMD, the defects were completely filled with BDX. The flaps were reposi- tioned and sutured.

Two animals from each treatment group were euthanized by intravenous injection of an overdose of sodium pentobarbital at 1, 2, 4, and 8 weeks after treatment. The jaw of each animal was removed, and specimens were decalcified and embedded in paraffin.

They were then stained with hematoxylin-eosin. From each root, five sec- tions were used for microscopic examination and histometric assessment.

Eight weeks after surgery, the following

Fig 1(left) A two-wall periodontal defect was prepared by removing bone with round burs and chisels.

Fig 2(right) Placement of composite graft of BDX plus EMD into defect.

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quantitative parameters were evalu- ated (Fig 3): apical notch to bone crest (new bone), apical notch to newly formed cementum (new cementum), and extent of junctional epithelium (epithelium). The percentage of each major tissue type filling the original defect (eg, new bone, new cementum, and epithelium) was calculated accord- ing to the following formula: the cross- sectional length of each tissue type was divided by the cross-sectional length of the original defect (apical notch to cementoenamel junction).

These measurements were statistically analyzed using the Scheffe test (n = 6) to determine whether the different treatments had any significant effect on the tested histometric parameters.

Results

Histologic observations

Combined treatment

After 1 week, the bone defects were filled with newly formed granulation tissue composed of fibroblasts and capillaries (Fig 4a). Most of the implanted BDX particles remained within the defects, and a large number

between the teeth and the newly formed bone (Fig 4h). Importantly, no marked root resorption or ankylosis was observed.

BDX alone

After 8 weeks some BDX particles at the bases of the defects were surrounded by new bone tissue, whereas other BDX particles in the middle and coronal parts of the defects were surrounded by dense connective tissue (Fig 5a). Newly formed cementum was present from the notch of the root surface to the coronal portion (Fig 5b).

Controls

After 8 weeks, the bone defect remained; a small amount of new bone had formed at its most apical portion.

Formation of new cementum was limited (Fig 5c). A long junctional epithelium extended apically along the entire length of the root surface (Fig 5d).

of spindle-shaped fibroblasts were interspersed throughout in a dense fibrillar extracellular matrix between the BDX particles (Fig 4b). After 2 weeks, the newly formed bone tissue had reached some of the implanted BDX particles (Fig 4c). Furthermore, new bone had been generated notice- ably around the BDX particles. The surfaces of the particles were surrounded by thin layers of bone tissue (Fig 4d). No new cementum formation was observed on the root surfaces.

After 4 weeks, new bone formation was found to have been initiated, with some BDX particles observed at the bases of the defects, surrounded by new bone tissue (Fig 4e). The most striking feature at this time was that the downgrowth of the junctional epithelium did not extend to the base of the defect. The layer of new cementum was thicker in the apical portion of the root surface and had a lining of cells resembling cementoblasts (Fig 4f).

After 8 weeks, most of the defects were completely filled with BDX particles, which were surrounded by new bone (Fig 4g). New bone and cementum formation were observed extend- ing along the coronal portion. A well- organized PDL space was observed

Fig 3 Histometric measurements. CEJ = cementoenamel junction; E = apical border of junctional epithelium; C = coronal level of newly formed cementum; B = coronal level of newly formed alveolar bone; N = apical border of notch.

CEJ E CB

N

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Fig 4 Histology of periodontal healing of defects that received the BDX+EMD treatment (hematoxylin-eosin). Squares indicate areas of higher magnification.

Figs 4a and 4b One week after surgery, the defect is filled with BDX particles and numerous fibroblasts (original magnification: a [left] ⫻5, b [right] ⫻100).

Figs 4c and 4d Two weeks after surgery.

Newly formed bone tissue reaches some of the BDX particles in the defects (original magnification: c [left] ⫻5, d [right] ⫻100).

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Figs 4e and 4f Four weeks after surgery.

The defect contains BDX particles, which are surrounded by new bone. Newly formed cementum extends from the apical notch (original magnification: e [left] ⫻3.1, f [right] ⫻100).

Figs 4g and 4h Eight weeks after surgery.

Newly formed bone is observed filling the defect area. A well-organized PDL is observed between the new cementum and the newly formed bone (original magnification: g [left] ⫻3.1, h [right] ⫻100).

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Fig 5 Histology of periodontal healing of the defects at 8 weeks after surgery (hematoxylin-eosin). Squares indicate areas of higher magnification.

Figs 5a and 5b Samples treated with BDX alone. BDX particles are surrounded by new bone at the base of the defect, while BDX particles in the middle and coronal parts of the defect are surrounded by dense connective tissue. Newly formed cementum is present in the middle parts of the root (original magnification: a [left] ⫻5, b [right] ⫻100).

Figs 5c and 5d Control defect. Defect is not completely filled with new bone. A long junctional epithelium is observed (original magnification: c [left] ⫻5, d [right] ⫻50).

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Histometric measurements

The histometric results at 8 weeks after surgery are shown in Table 1. The BDX+EMD group showed an increase in new cementum of 89.8% ± 11.7%

(ie, 4.5 mm) and in new bone of 52.2%

± 21.7% (ie, 3.6 mm). In contrast, in the BDX group, new cementum and new bone increased by 45.0% ± 18.1% (ie, 2.9 mm) and 31.6% ± 9.6% (ie, 2.0 mm), respectively. This demonstrated that the growth of new cementum and new bone in the BDX+EMD group was significantly higher (P < .05) than that in the BDX-only group. The BDX+EMD group showed a 21.7% ± 1.4% (ie, 1.4 mm) increase in the mean length of the epithelium, which was not a significant difference from that seen in the BDX-only group (20.6% ± 1.9%, ie, 1.8 mm). However, the BDX+EMD group (1.4 mm) showed a significantly lower mean length of epithelium than the control group (2.9 mm) (P < .01).

Discussion

Histologically, significant improve- ments were noted in both the BDX+EMD and the BDX-only groups compared to the control group. A comparison of the BDX+EMD and BDX- only treatment groups revealed that more new cementum and bone were produced in the BDX+EMD group, indicating that the combined treatment offered a stronger effect. The results of this study support the hypothesis that therapy combining BDX and EMD induces periodontal regeneration in large intrabony defects.¹⁹In the control group, only a small amount of periodontal tissue regeneration was noted near the notch on the root surface, suggesting that spontaneous healing is difficult when wider bone defects (5 ⫻ 5 ⫻5 mm) are involved. This suggests that, with spontaneous healing, prolif- erating soft tissues enter the bone defect, thus preventing proliferation of undifferentiated cells and soft tissue containing growth factors derived from the surrounding periodontal tissues during the wound-healing process.

This has previously been shown in large

Table 1 Histometric parameters (means and standard deviations [%] and means [mm]) for each surgical treatment (n = 8) at 8-week sites

Means ± standard deviations (%) and means (mm)

Tissue BDX+EMD BDX alone Control

New cementum 89.8 ± 11.7 (4.5 mm)*^,** 45.0 ± 18.1 (2.9 mm)* 16.2 ± 1.29 (1.4 mm)**

New bone 52.2 ± 21.7 (3.6 mm)*^,** 31.6 ± 9.6 (2.0 mm)* 18.2 ± 2.51 (1.2 mm)**

Epithelium 21.7 ± 1.4 (1.4 mm)^NS,** 20.6 ± 1.9 (1.8 mm)^NS 55.3 ± 0.9 (2.9 mm)**

*P< .05 BDX+EMD versus BDX alone; **P< .01 BDX+EMD versus control; NS = not significant.

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bone defects in dogs; that the greater the distance to the adjacent bone wall, the smaller the influence of the natural bone.²¹

Histologic examination revealed no epithelial downgrowth in the BDX+EMD group or in the BDX group, whereas a long epithelial attachment was shown in the control group. The rate of epithelial downgrowth was significantly lower in the BDX+EMD group and in the BDX-only group compared to the control group (1.4 mm and 1.8 mm vs 2.9 mm; P < .01). BDX plus EMD treatment or BDX alone showed new connective tissue attach- ments occurring at the root surface when PDL cells were allowed to cover those surfaces prior to the arrival of epithelial cells. It has been proposed that inhibition of epithelial downgrowth promotes the regeneration of periodontal tissues, including new cementum and bone.^22,23

In this study, the rate of new bone formation in the wider bone defects in the BDX-only group was about 1.7 times (2.0 mm) that seen in the control group (1.2 mm). This suggests that bone grafting materials are useful scaf- folds for periodontal tissue regeneration in various periodontal bone defects.²⁴Klinge et al²⁵reported that BDX was an ideal scaffold for the regeneration of new cementum and bone in experimental bone defects in rabbits. BDX seems to promote bone formation earlier than other graft materials. In this study, histologic examination confirmed the positive osteocon- ductive properties of BDX, as documented by the close contact between this material and the newly formed bone. However, bone regen-

eration was lower in the BDX-only group than in the BDX+EMD group (2.0 mm vs 3.6 mm; P < .05). In the present study, histologically, BDX was mostly embedded in fibrous connective tissue, which was distant from the alveolar bone. This may have been caused by the difficulty of applying BDX evenly in such large bone defects.

A more even distribution might enable optimal stabilization of blood coagu- lation, thus providing the necessary scaffold for new bone formation.²⁴

EMD-induced new bone and cementum formation is affected by the interaction between the bone conductivity of BDX and the cementum conductivity of EMD. When BDX and EMD were applied together to periodontal intrabony defects, periodontal tissue regeneration was increased.¹⁹ In this study, the amount of new cementum formed in the EMD+BDX group was two times higher than that in the group treated with BDX alone (4.5 mm vs. 2.9 mm; P < .05). In terms of bone regeneration, the amount of new bone formed in the BDX+EMD group was 1.7 times higher than that in the group treated with BDX alone.

The viscosity of polyglycolic acid assists in the delivery of BDX particles by holding them together, thus facilitating their distribution throughout the defect. It has also been speculated that the superior outcome of the combined treatment approach is a result of the enhanced blood clot stabilization in bony defects and the isolation of gingival epithelial and connective tissue cells from the defect areas.²⁶The biologic mechanism underlying the induction of EMD activity has yet to be elucidated. EMD mimics the early

stage of root development, thus form- ing an environment on the root surface that stimulates the development of acellular cementum, PDL, and bone.²⁷ It is believed that EMD allows the appropriate development of attach- ments through the action of cementoblasts and osteoblasts.²⁸The function of polyglycolic acid plus EMD as a matrix is to form a scaffold for cell proliferation and differentiation and to provide suitable conditions for matrix production. In wider periodontal bone defects, where the supply of appropriate PDL cells from periodontal tissue is enriched, EMD alone may be enough to facilitate the osteoconduc- tive properties of BDX.

The results of this study suggest that the use of EMD, which is an osteo- promotive agent, in combination with BDX increases the bone conductivity of graft materials. This histologic inves- tigation has demonstrated that the combination of BDX and EMD significantly enhances periodontal regeneration in wider bone defects, suggesting that this type of treatment is favorable to flap surgery alone.

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Acknowledgments

This study was supported by grants from the Japanese Ministry of Education, Science and Culture (no. 10470460).

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