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Journal of Indian Society of Pedodontics and Preventive Dentistry Official publication of Indian Society of Pedodontics and Preventive Dentistry
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ORIGINAL ARTICLE
Year : 2013  |  Volume : 31  |  Issue : 4  |  Page : 249-253
 

Vitapex can promote the expression of BMP-2 during the bone regeneration of periapical lesions in rats


1 Department of Dentistry, The Second Affiliated Hospital of Harbin Medical University, Harbin, China
2 Laboratory of Medical Genetaics, Harbin Medical University, Harbin, China

Date of Web Publication21-Nov-2013

Correspondence Address:
Xiumei Wang
Department of Dentistry, the Second Affiliated Hospital of Harbin Medical University, 246 Xuefu Road, Nangang District, Harbin 150086
China
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Source of Support: This work was supported by Natural Science Foundation of Heilongjiang Province of China (D201175), and Postdoctoral Science Foundation of Heilongjiang Province (LRB05-279) (to X.W.),, Conflict of Interest: None


DOI: 10.4103/0970-4388.121825

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   Abstract 

Purpose: To investigate the effect of Vitapex on the healing of periapical lesions and the expression of bone morphogenetic protein (BMP-2) during the periapical bone regeneration. Materials and Methods: Periapical lesions were induced in Sprague-Dawley (S-D) rats by an occlusal pulp exposure in the mandibular first molars and were verified by X-ray. Total of 36 rats were randomly divided into three groups, and they were obturated with Zinc Oxide Eugenol (ZOE), or with Vitapex, or non-treated as negative control group. The rats of three groups were randomly killed at week 0, 2, 4, and 8 after root canal therapy, and then the mandibles were processed for histological examination and immunohistochemistry analysis. Results: At week 0, only a few BMP-2 positive cells could be observed in all rats. While the expression of BMP-2 was dramatically increased in case of Vitapex group at week 2 and week 4, and then climaxed at week 8. However, no apparent changes were observed in ZOE group and negative group at week 2, 4, and 8. Conclusion: These observations suggested that Vitapex has a greater ability in inducing bone regeneration than ZOE by the expression of BMP-2 induction in the treatment of rats experimental periapical lesions.


Keywords: Apical periodontitis, BMP-2, bone regeneration, Vitapex


How to cite this article:
Xia X, Man Z, Jin H, Du R, Sun W, Wang X. Vitapex can promote the expression of BMP-2 during the bone regeneration of periapical lesions in rats. J Indian Soc Pedod Prev Dent 2013;31:249-53

How to cite this URL:
Xia X, Man Z, Jin H, Du R, Sun W, Wang X. Vitapex can promote the expression of BMP-2 during the bone regeneration of periapical lesions in rats. J Indian Soc Pedod Prev Dent [serial online] 2013 [cited 2019 Nov 16];31:249-53. Available from: http://www.jisppd.com/text.asp?2013/31/4/249/121825



   Introduction Top


Apical periodontitis is an infectious disease caused by microorganisms, mainly bacteria that colonize the necrotic root canal system and invade periapical tissues. [1] The inflammatory process ultimately results in periapical bone resorption. [2],[3] Vitapex is widely used in the preparations for obturation of primary tooth pulpectomies, and gives excellent results when used as an obturating material in apical periodontitis. The material shows (i) good resorbable properties, (ii) is radiopaque in nature, (iii) does not set to a hard mass, (iv) is easily inserted and removed from the canals. [4],[5] Vitapex has the effect of sustained anti-bacterial activity and can effectively inhibit the propagation of apical pathogens. Studies have supported that the observation of long-term application of Vitapex can affect the metabolism of the periapical bone tissue and can induce new bone tissue regeneration and promote the healing of periapical lesions when extruded beyond the apex of the tooth when used as an obturating matetial. [6]

Bone morphogenetic proteins (BMPs) were originally isolated from bone tissues as molecules that induce bone and cartilage formation in ectopic extra-skeletal sites in vivo.[7] BMPs have been investigated in the delayed reconstruction of bone defects secondary to open fracture. The rationale for their use in this setting is based on the concept that BMPs combined with an osteoconductive substance can promote healing of bone defects in a fashion comparable to that of autogenous bone graft while avoiding the complications and limitations associated with autogenous bone grafting. The positive results of animal studies have provided the basis for the clinical investigation of BMPs in the healing. Especially BMP-2 are known for osteoinductive qualities. [8],[9],[10] BMP-2 is one of the most critical factors that mainly promote differentiation of undifferentiated mesenchymal cells to osteogenic lineage cells in early process of bone formation. [11] As other parts of the bone formation in body, BMP-2 is the initial participant in the development of periapical bone formation. [12]

Vitapex is used as root canal obturating paste, on the contrary, it inhibits or kills the pathogen in the root canal; further, it can promote bone repairment and regeneration. [6] Therefore, we hypothesize that Vitapex may promote BMP-2 expression of periapical lesions to improve periapical bone regeneration. To our knowledge, there has been no report on the expression of BMP-2 induced by Vitapex in periapical lesions. This study aims to examine the expression of BMP-2 and discuss its relationship with Vitapex at different stages of periapical lesions in rats.


   Materials and Methods Top


Induction of Periapical Lesions and radiographic examination

A total number of 36 female Sprague-Dawley (S-D) rats weighing 250-270 g were purchased from the Experimental Animal Center of Harbin medical university (Harbin, China). Periapical lesions were induced in rats by occlusal exposure of the lower first molar pulps with the method described by Stashenko et al., [13] and Wang et al., [14] Under ketamine (90 mg/kg, intraperitoneal) anesthesia, an occlusal class I cavity was made with a half-round bur, which penetrated to a depth equal to the diameter of its active part. The pulps were then left open to the oral environment. Periapical lesions were verified by X-ray. These rats were treated with conventional root canal therapy. After root canal preparation and irritation, they were divided into three groups. The first group was treated with ZOE as root obturating materials, the second group was treated with Vitapex as root obturating paste, and the third group was not treated with anything. After root canal therapy, the borders of the radiographic image of each periapical lesion were traced and measured at week 0, 2, 4, and 8.

Immunohistochemistry analysis

Three rats of each group were killed on week 0, 2, 4, and 8 after root canal therapy. The mandibles were fixed with 4% paraformaldehyde in 0.1 mol/L phosphate-buffered saline (PBS) (pH 7.4) at 4°C for 48 hours. After that, the specimens were decalcified with 10% ethylenediaminetetraacetic acid at 4°C for 4 weeks. Next, the specimens were dehydrated in an ethanol series and embedded in paraffin. Serial sections were cut at a thickness of 5 mm. The sections that included the distal root of the first mandibular molars and exhibiting a patent root canal apex representing the central portion of root canal were selected for histologic and immunohistochemical analysis. The streptavidin-peroxidase (SP)-conjugated method was applied with the anti-BMP-2 antibodies (Boshide Biotechnology Co, Wuhan, China) for 1 hour at 37°C, and stained with SP kit (Zhong shan Biotechnology Co, Beijing, China) according to the manufacturer's instructions. Tissue samples of normal rat root apex were used as negative control. Counterstaining was conducted with hematoxylin. In each specimen, BMP-2 positive cells in the periapical tissues were counted under high-power magnification (×400). A technician randomly picked up five visual fields in the area and counted cells according to the single blind principle. The average number per high-power field in each group was then subjected to statistical analysis with one-way analysis of variance and Pearson correlation at a = 0.05 by using the Statistical Package for Social Sciences (SPSS) version 13.0.


   Results Top


Radiographic examination showed the absorption of the paste of Vitapex was evident

After root canal therapy, in different experimental periods the X-ray irradiation showed that the differences of paste absorption and transmission of periapical tissue among the three groups, especially the changes of Vitapex group is significant. The borders of the radiographic image of each periapical lesion were traced and calculated at week 0, 2, 4, and 8. In Vitapex group, the paste is absorbed more than the ZOE group at week 2 and 4, and further narrowing the scopes of periapical lesions. At week 8, the absorption of Vitapex was evident than the ZOE group and the negative group, as well as narrowing the scopes of periapical lesions [Figure 1] a-h.
Figure 1: The X-ray irradiation showed the differences of paste absorption and transmission of periapical tissue among the three groups in different experimental periods. Vitapex group: a (At week 0), b(At week 2), c(At week 4), d( At week 8); ZOE group: e (At week 0), f (At week 2), g (At week 4), h ( At week 8)

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The expression of BMP-2 is significantly increased with Vitapex treatment

Immumoreactivity is performed on each group after root canal therapy on week 0, 2, 4, and 8. All of three groups, a few BMP-2 positive cells could be observed at week 0 after root canal therapy (P > 0.05). The expression of BMP-2 positive cells in Vitapex group increased dramatically at week 2 and 4; however, there was no apparent changes in ZOE group and negative group at week 2 and 4 (P < 0.05). The expression of BMP-2 positive cells in vitapex group climaxed at week 8, whereas there was also no apparent changes in ZOE group and negative group at week 8 (P < 0.01) [Figure 2] a-l, [Table 1].
Table 1: The rate of BMP-2 in rats periapical lesions at different time after root canal therapy (x±s)

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Figure 2: The expression of BMP-2 in rats periapical lesions (SP×400). Negative group: a (At week 0), b (At week 2), c (At week 4), d ( At week 8); Vitapex group: e (At week 0), f (At week 2), g (At week 4), h ( At week 8); ZOE group: i (At week 0), j (At week 2), k (At week 4), l ( At week 8)

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   Discussion Top


Chronic apical periodontitis is initially treated with conventional root canal treatment agents and irrigants, then the apical inflammatory granulation tissue will be into fibrous connective tissue and the active osteoblasts that will produce new bone, repair the undermined alveolar bone and reconstruct the periodontal ligament. [15],[16] The current study suggests that in the bone regeneration process, one of the most closely related factors is BMP-2. The BMP-2 is an important induced differentiation factor which plays a major role in the embryonic development and tooth tissue formation and adult bone repairmen. [17] Use of limb-specific targeted disruption of individual BMPs has defined an essential role for BMP-2 in the postnatal skeleton. [18],[19] In vitro with recombinant human bone morphogenetic protein-2 (rhBMP-2), therapeutic agent for treatment bermudagrass periapical disease experiments confirmed that BMP-2 can promote undifferentiated mesenchymal cells to differentiate into osteoblasts, ascementum, and fibroblast cells; and enhance the formation of the alveolar bone, cementum, and periodontal ligament; and reconstruct the normal periapical tissue. [20]

It can be observed from the experimental results that Vitapex group, the ZOE group or negative group periapical lesions area has the distinct expression of BMP-2. This indicates that BMP-2 involved in the apical lesions area of bone regeneration process. It was observed that BMP-2 positive cells in Vitapex group were significantly increased, while ZOE group and negative group have a slight expression of BMP-2. The increased expression of BMP-2 in Vitapex group may be related to the local effects of Vitapex. Analysisat week 0, 2, 4, and 8 periods of three groups of BMP-2 expression, it was observed that the Vitapex group showed a trend from low to high expression, while ZOE group and negative group did not show any significant change, suggesting that Vitapex has the sustained effect on healing of periapical bone destruction. From [Figure 1], it was observed that the paste of Vitapex group was absorbed significantly and the scope of bone destruction of Vitapex group was apparently reduced which matches the trend of the expression of BMP-2. Vitapex has the role of promoting the periapical bone regeneration in vivo environment and this effect may be related to the rise of local BMP-2 expression level.

The main ingredient of Vitapex are iodoform, calcium hydroxide, and polysiloxane oil. [21] Calcium hydroxide is strongly alkaline and can kill bacteria inside the root canal. Further it can induce the capacity of the hard tissue formation and promote regeneration of cementum, and promote periapical tissue calcification. Calcium hydroxide being a strong alkaline can neutralize the acid products of inflammatory cells and enhance alkaline phosphatase activity. It plays the role of extending roots and apical closure. Polysiloxane oil is fluidity and permeability, the collateral benefit root canal filling. [22],[23] Iodoform releases free iodine when meeting tissue fluid, with a strong long-lasting disinfection and can also eliminate the infection of root canal and periapical tissue. [24],[25] In this study, Vitapex have the effective role of inducing bone formation of apical periodontitis and the expression of BMP-2 increased more than other groups.


   Conclusion Top


Vitapex has the specific bone regeneration effect of removing inflammatory granulation tissue and this effect may be related to the rise of local BMP-2 expression level.

 
   References Top

1.Zhang X, Peng B. Immunolocalization of receptor activator of NF kappa B ligand in rat periapical lesions. J Endod 2005;31:574-7.   Back to cited text no. 1
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2.Kawashima N, Stashenko P. Expression of bone-resorptive and regulatory cytokines in murine periapical inflammation. Arch Oral Biol 1999;44:55-66.  Back to cited text no. 2
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3.Stashenko P, Teles R, D'Souza R. Periapical inflammatory responses and their modulation. Crit Rev Oral Biol Med 1998;9:498-521.  Back to cited text no. 3
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6.Gu HJ, Xu Q, Liu LM, Quyang Y. Treatment of chronic apical periodontitis teeth complicated by open apices with Vitapex in the adults. Shanghai Kou Qiang Yi Xue 2007;16:140-3.  Back to cited text no. 6
    
7.Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, et al. Novel regulators of bone formation: Molecular clones and activities. Science 1988;242:1528-34.  Back to cited text no. 7
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21.Nakornchai S, Banditsing P, Visetratana N. Clinical evaluation of 3Mix and Vitapex as treatment options for pulpally involved primary molars. Int J Paediatr Dent 2010;20:214-21.  Back to cited text no. 21
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