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DENTAL SCIENCE - ORIGINAL ARTICLE
Year : 2013  |  Volume : 5  |  Issue : 5  |  Page : 48-53  

Effect of various concentrations of tetracycline hydrochloride demineralization on root dentin surface: A scanning electron microscopic study


1 Department of Periodontics, Gitam Dental College and Hospital, Vishakhapatnam, Andhra Pradesh, India
2 Department of Orthodontics, Gitam Dental College and Hospital, Vishakhapatnam, Andhra Pradesh, India
3 Department of Periodontics, R.M.D.C and H., Chidambaram, Tamil Nadu, India
4 Department of Prosthodontics, Gitam Dental College and Hospital, Vishakhapatnam, India

Date of Submission02-May-2013
Date of Decision04-May-2013
Date of Acceptance04-May-2013
Date of Web Publication13-Jun-2013

Correspondence Address:
Tanuja Penmatsa
Department of Periodontics, Gitam Dental College and Hospital, Vishakhapatnam, Andhra Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0975-7406.113296

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   Abstract 

Introduction: Within the last 20 years root demineralization has attracted attention as a periodontal regenerative technique. Topical tetracycline application has been widely reported for use as a conditioner, to decontaminate the root surface and to promote periodontal regeneration. The purpose of this scanning electron microscopic (SEM) study is to evaluate the surface characteristics of demineralized diseased and non-diseased dentine root surfaces using different concentrations of tetracycline hydrochloride (TTC-HCl). Material and Methods: This SEM study was carried out in 20 dentin samples obtained from non-diseased human premolars and 20 dentin samples obtained from diseased human premolars. Pure TTC-HCl was applied to the dentin surface at 50 mg/ml, 100 mg/ml and 150 mg/ml concentrations for 3 min in experimental groups while distilled (0 mg/ml) water was applied to the control groups. Statistical Analysis: Mean and standard deviation were estimated from the sample for each study group. Mean values were compared by either one-way ANOVA with Tukey-Honestly significant difference procedure or Kruskal - Wallis one way ANOVA with Mann-Whitney u-test. Further Student's independent t-test/Mann-Whitney u-test was used appropriately to compare the mean values between two independent groups. Results: In the present study using 100 mg/ml tetracycline seems to be more effective on both diseased and non-diseased dentin surfaces. Conclusions: The results of this study confirm that pure TTC-HCl conditioning produced comparable surface characteristics on dentin of both diseased and non-diseased roots with 100 mg/ml concentration for 3 min.

Keywords: Dentine surfaces, demineralization, root conditioning, tetracycline hydrochloride


How to cite this article:
Penmatsa T, Varma S, Mythili, Rao KP, Kishore T, Bindu H. Effect of various concentrations of tetracycline hydrochloride demineralization on root dentin surface: A scanning electron microscopic study. J Pharm Bioall Sci 2013;5, Suppl S1:48-53

How to cite this URL:
Penmatsa T, Varma S, Mythili, Rao KP, Kishore T, Bindu H. Effect of various concentrations of tetracycline hydrochloride demineralization on root dentin surface: A scanning electron microscopic study. J Pharm Bioall Sci [serial online] 2013 [cited 2022 Nov 30];5, Suppl S1:48-53. Available from: https://www.jpbsonline.org/text.asp?2013/5/5/48/113296

One of the objectives of periodontal therapy is the restoration of the lost periodontium and conversion of the periodontitis - affected root surface into a substrate which is biologically hospitable for epithelial and connective tissue cell adherence and attachment. Within the last 20 years, root demineralization has attracted attention as a periodontal regenerative technique. [1],[2]

Adequate removal of plaque, calculus and cytotoxic substances from the diseased root surface appears to be essential for periodontal regeneration. [3] In addition, the dentin surface smear layer [4],[5] produced by most forms of root manipulation could potentially affect fibroblast adaption in the healing periodontal wound. A number of agents have been used in conjunction with demineralization, including, phosphoric acid, Ethelene Diamine Tetra Acetic Acid (EDTA), citric acid [6],[7] and tetracycline. [8] Topical tetracycline application has been widely reported for use as a conditioner, to decontaminate the root surface and to promote periodontal regeneration.

The tetracyclines are a group of bacteriostatic antimicrobials effective against a wide range of organisms. Concentrated tetracycline hydrochloride (TTC-HCl) solution is moderately acidic (pH 1-2) and removes the surface inorganic smear layer, created on the tooth during most dental treatments to expose underlying dentin and tubules [9],[10],[11],[12]

Tetracycline studies demonstrate multiple beneficial properties towards regeneration, enhanced attachment and growth of gingival fibroblasts; good anti-collagenase activity, high substantivity; inhibition of parathyroid hormone - induced bone resorption and anti-inflammatory action. [13],[14] In addition, it has been indicated that tetracycline is absorbed and subsequently desorbed from dentin, maintaining its antimicrobial activity. [15],[16],[17] These findings have led to widespread use of tetracycline treatment on root surfaces in periodontal therapy.

The purpose of this study is to evaluate the surface characteristics of demineralized diseased and non-diseased dentin surfaces using different concentrations of TTC-HCl.


   Materials and Methods Top


This in vitro study includes the following groups.

  1. Study I → control group (A) and experimental groups (B, C and D).
  2. Study II → control group (E) and experimental groups (F, G and H).
In the study I, the surface characteristics of the demineralized diseased root dentin surface using various concentration of TTC-HCl with 3 min application period were compared. In study II the surface characteristics of the demineralized non-diseased root dentin surface using various concentration of TTC-HCl with 3 min application period were compared. Each group in study I and study II had five dentin samples (total 40 samples).

20 dentin blocks were prepared from non-diseased human premolars [18] which were extracted for orthodontic reasons, and 20 dentin blocks were obtained from diseased premolars, [16] which were extracted due to advanced periodontitis. Care was taken that all the selected patients were 15-45 years age group and the teeth were devoid of root caries or restorations. The diseased tooth had attachment loss of more than 6 mm.

Immediately after extraction loose debris/calculus was removed with a hand scaler, and the teeth were cleaned and rinsed in distilled water. The root is then planed thoroughly using a curette and a fine diamond tapered fissure bur. Anatomical crown and apical third of the root was removed and mid-root of the non-diseased and diseased tooth was used to prepare the dentin blocks. The specimens were sectioned longitudinally through the root canal using a high speed hand piece with copious water and a tapered fissure bur.

The pulpal tissue was removed and a horizontal shallow groove for identification purpose was made on the pulpal surface of each specimen. Immediately thereafter each root surface was thoroughly planed and flattened with a fine diamond tapered fissure bur to completely remove cementum and expose the underlying dentin. The dentin blocks were divided into 8 groups (4 diseased and 4 non-diseased) having five samples in each group.

Preparation of TTC-HCl solutions

Fresh TTC-HCl solutions were prepared at room temperature by weighing pure TTC-HCL (FLUKA) and dissolving in 10 ml of distilled water, using a glass rod to mix the solution. The concentrations, of the TTc-HCl test solutions were 50 mg/ml, 100 mg/ml and 150 mg/ml. The control groups group A and group E were treated with distilled water. The study groups B and F were conditioned with 50 mg/ml TTc-HCl solution, Group C and G were conditioned with 100 mg/ml and Group D and group H were conditioned with 150 mg/ml TTC- HCl solutions.

The specimens in each group were conditioned for 3 min with a solution soaked cotton pellet. The cotton pellet is changed every 30 s. After 3 min dentin surface was flushed with distilled water for 30 s to stop the chemical reaction.

Preparation for scanning electron microscopic (SEM)

Immediately after conditioning with TTC-HCl and rinsing, the dentin blocks were placed in 10% formalin overnight and then dehydrated in a graded series of iso-propyl alcohol for 10 min each. The samples were then dried and placed in a dessicator jar. The dentin blocks were then mounted on brass stubs and sputter coated with platinum for 2 min in sputter coater (JFC - 1600 from JEOL). The coated surfaces were then scanned and observed on the monitor at a magnification of × 1500 (JEOL JSM - 5610 LV SEM).The following surface characteristics were studied for each specimen.

  1. Efficacy of removal of smear layer.
  2. Total number of dentinal tubules exposed
  3. Diameter of selected 10 dentinal tubules.
Statistical analysis

Mean and standard deviation were estimated from the sample for each study group. Mean values were compared by either one-way ANOVA with Tukey-Honestly significant difference procedure or Kruskal - Wallis one way ANOVA with Mann-Whitney u-test. Further Student's independent t-test/Mann-Whitney u-test was used appropriately to compare the mean values between two independent groups. In the present study P < 0.05 was considered as the level of significance.

There is no significant difference in smear layer removal between the study groups of diseased dentin surface using 50 mg/ml, 100 mg/ml and 150 mg/ml. Mean smear layer score in Group B (3.8 ± 0.4), Group C (4.0 ± 0.0) and Group D (4.0 ± 0.0) are significantly higher than Group A (0.0 ± 0.0 (P < 0.05). Mean number of exposed dentinal tubules in Group C (81.2 ± 1.9) was significantly higher than Group A (10.8 ± 0.8), Group B (38.0 ± 3.8) and Group D (70.6 ± 6.8) (P < 0.05). Mean diameter of the dentinal tubules in Group C (3.87 ± 0.04) is significantly higher than Group A (0.93 ± 0.06), Group B (2.30 ± 0.04) and Group D (3.57 ± 0.12) (P < 0.05).

Mean number of exposed dentinal tubules using 100 mg/ml was significantly higher than 0 mg/ml, 50 mg/ml, and 150 mg/ml [Figure 1], [Figure 2], [Figure 3] and [Figure 4]. Mean diameter of the dentinal tubules using 100 mg/ml is significantly higher than that of using 0 mg/ml, 50 mg/ml, 150 mg/ml [Figure 1], [Figure 2], [Figure 3] and [Figure 4]. Mean smear layer score in group F (3.6 ± 0.5), Group G (4.0 ± 0.0) and group H (4.0 ± 0.0) are significantly higher than Group E (0.0 ± 0.0) (P < 0.05). There was no significant difference in mean smear layer score between other contrasts (P > 0.05).
Figure 1: Scanning electron microscopic photograph of diseased dentin surface treated with distilled water

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Figure 2: Scanning electron microscopic photograph of diseased dentin surface treated with 50 mg/ml Tetracycline Hydrochloride solution

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Figure 3: Scanning electron microscopic photograph of diseased dentin surface treated with 100 mg/ml Tetracycline Hydrochloride solution

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Figure 4: Scanning electron microscopic photograph of diseased dentin surface treated with 150 mg/ml Tetracycline Hydrochloride solution

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Mean smear layer removal in the study group using concentration 50 mg/ml, 100 mg/ml, 150 mg/ml on non-diseased dentin surface was significantly higher than the control (0 mg/ml). However, there was no significant difference in mean smear layer score between other contrasts (using 50, 100, 150 mg/ml) [Figure 5], [Figure 6], [Figure 7] and [Figure 8]. Mean number of exposed dentinal tubules in group G (79.2 ± 1.9) was significantly higher than Group E (10.4 ± 0.5), Group F (36.0 ± 2.0) and Group H (71.6 ± 3.6) ( P < 0.05).
Figure 5: Scanning electron microscopic photograph of non-diseased dentin surface treated with distilled water

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Figure 6: Scanning electron microscopic photograph of non-diseased dentin surface treated with 50 mg/ml Tetracycline Hydrochloride solution

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Figure 7: Scanning electron microscopic photograph of non-diseased dentin surface treated with 100 mg/ml Tetracycline Hydrochloride solution

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Figure 8: Scanning electron microscopic photograph of non-diseased dentin surface treated with 150 mg/ml Tetracycline Hydrochloride solution

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Mean number of exposed dentinal tubules using 100 mg/ml concentration of TTC was significantly higher than using 150 mg/ml, 50 mg/ml, 0 mg/ml [Figure 5], [Figure 6], [Figure 7] and [Figure 8]. Mean diameter of the dentinal tubules in Group G (3.87 ± 0.03) was significantly higher than Group E (0.94 ± 0.03), Group F (2.26 ± 0.05) and Group H (3.28 ± 0.06) ( P < 0.05). Mean diameter of the dentinal tubules using 100 mg/ml on non-diseased dentin surface was significantly higher than using 150 mg/ml, 50 mg/ml and 0 mg/ml [Figure 5], [Figure 6], [Figure 7] and [Figure 8]. No comparison was found between mean values of smear layer scores between the diseased and non-diseased dentin surfaces [Table 1]. No significant difference was found between diseased and non-diseased dentin surfaces using different concentrations of TTC-HCl in the exposure of the number of dentinal tubules [Table 2]. Mean diameter of dentinal tubules in Group D (3.57 ± 0.12) was significantly higher than Group H (3.28 ± 0.06) ( P = 0.001). The mean diameter of the dentinal tubules using 150 mg/ml of tetracycline on diseased dentin surface was significantly higher than the non-diseased dentin surface. However, there was no difference in the mean diameter of the dentinal tubules between diseased and non-diseased dentin surfaces using tetracycline in 100 mg/ml and 50 mg/ml [Table 3].
Table 1: Comparison of mean values of smear layer scores between diseased (study I) and non-diseased dentin surfaces (study II)

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Table 2: Comparison of mean values of exposed dentinal tubules between diseased (study I) and non-diseased (study II) dentin surface

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Table 3: Comparison of mean values of the diameter of the dentinal tubules between diseased (study I) and non-diseased (study II) dentin surfaces

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   Discussion Top


It is well established that the periodontally diseased root surface does not favour regeneration of periodontium due to surface characteristics. One of the objectives of periodontal therapy is to convert the periodontitis affected root surfaces into a substrate which is biologically compatible for epithelial and connective tissue adherence and attachment. Methods to achieve this objective have included mechanical treatment methods like scaling and root planing and the use of some demineralization agents. Instrumentation of the root surfaces, however, results in the formation of a smear layer of organic and mineralized debris. [5],[10] This smear layer is thought to serve as a physical barrier between the periodontal tissues and root surfaces and may inhibit the formation of the connective tissue attachment to the root surface. TTC-HCl conditioning of the root surface not only will selectively remove the surface smear layer, but may also act favorably by inhibiting collagenase activity and bone resorption and by its local antimicrobial effects. [9]

Baker et al., [15] in their study showed that TTC-HCl and its derivatives strongly adsorb to tooth surfaces retaining their antimicrobial activity. The tooth surfaces release active antibiotic, as well as, serve as an improved substrate for connective tissue components vital to healing. Madison et al. [19] compared the effects of different tetracycline's on dentin root surface and suggested that TTC-HCl is the best current tetracycline form for root surface conditioning as measured by its ability to affect both dentin smear layer removal and dentin tubule exposure.

In this study, control groups A and E which were treated with distilled water (0 mg/ml) showed the presence of smear layer, loose debris and the presence of depressions corresponding to dentinal tubule orifice. [14] The TTC-HCl concentrations used in this study were chosen based on the results of in vitro investigations. In these studies, removal of the smear layer with exposure of a collagenous matrix was observed when conditioned with concentrations of ≥ 50 mg/ml TTC-HCl. [11],[20],[21],[22]

The experimental groups of both diseased and non-diseased dentin surfaces showed a different picture. Smear layer removal was significantly higher with concentrations of 50 mg/ml, 100 mg/ml and 150 mg/ml than that of the controls i.e., 0 mg/ml in both diseased and non-diseased dentin surfaces.

In the present study, mean number of exposed dentinal tubules and mean diameter of the dentinal tubules using 100 mg/ml was significantly higher than 0 mg/ml, 50 mg/ml and 150 mg/ml. This finding was similar in both diseased and non-diseased dentin surfaces. Statistically significant difference was not found when the mean values of smear layer scores were compared between the diseased (Study I) and non-diseased (Study II) dentin surfaces.

The mean diameter of dentinal tubules using 150 mg/ml of TTC-HCl on diseased (Study I) dentin surface was significantly higher than the non-diseased (Study II) dentin surface. However, there was no difference in the mean diameter of the dentinal tubules between diseased and non-diseased surfaces using tetracycline 100 mg/ml and 50 mg/ml.

Pure tetracytine HCl was used for demineralizing the dentin surfaces as used by Isik et al. [2] as tetracycline capsules introduced filler particles and other substances into the solution. In the present study, cotton pledges soaked in TTC-HCl solution were applied for 3 min at each dentin surface [2],[6],[9],[23] In the present study, the TTC-HCl solution was applied using a burnishing techniques. It has been shown that the acid burnishing technique resulted in a chemical/mechanical action that enhances the removal of chemically loosened inorganic material and surface debris, exposing the underlying root surface to the demineralization action of fresh acid solution. This may ultimately achieve an optimal degree of demineralization within a short period of time, in comparison to other application modes. The present results regarding TTC-HCl concentrations showed that the differences in tubule diameters and exposure of the dentinal tubules were statistically significant between 50 mg/ml, 100 mg/ml and 150 mg/ml TTC-HCl. Although using 100 mg/ml tetracycline seems to be more effective on both diseased and non-diseased dentin surfaces.

In recent studies, different results have been reported in demineralization produced by various concentrations. Sterret et al. [23] found that higher concentrations of TTC-HCl demineralize dentin more effectively, which tend to leave more tetracycline on the root surface. They also demonstrated that tetracycline substantivity and desorbtion from the root surface was concentration dependent. In another study, greater number of attached cells was found at 100 mg/ml TTC-HCl concentration compared to untreated controls. Register and Burdick's [6],[24] findings suggested that initial and surface layer changes in the organic matrix caused by demineralization or subsequent cellular contact are important and that the over-application of the acid may disrupt these early events in the healing process. This supports the motion that exposure period of dentin to acid solution is critical and should be carefully established.

The results of this study confirm that pure TTC-HCl conditioning produced comparable surface characteristics on dentin of both diseased and non-diseased roots with 100 mg/ml concentration for 3 min.


   Conclusions Top


The present in vitro study evaluated the surface characteristics of demineralized diseased and non-diseased dentin surfaces using different concentrations of TTC-HCl for 3 min. From the present study, the following conclusion can be made.

  1. The dentin surfaces in control groups A and E were covered by a homogeneous smooth smear layer.
  2. Various concentrations used in this study were equally effective in removing the smear layer.
  3. 50 mg/ml, 100 mg/ml and 150 mg/ml concentrations of TTC-HCl exposed the dentinal tubules, but the number of dentinal tubules exposed was significantly more with 100 mg/ml tetracycline concentration.
  4. The diameter of dentinal tubules exposed by 100 mg/ml TTC-HCl concentration was statistically high as when compared to other concentrations. This finding was similar for both diseased and non-diseased dentin surfaces.
In view of these present findings, 100 mg/ml TTC-HCl as a root conditioner may be evaluated in-vivo studies. It is reasonable to expect lesser tissue destruction and better regeneration of periodontal tissues if it is used as a root conditioner during periodontal regeneration procedures.

 
   References Top

1.Polson AM, Caton J. Factors influencing periodontal repair and regeneration. J Periodontol 1982;53:617-25.  Back to cited text no. 1
    
2.Isik AG, Tarim B, Hafez AA, Yalçin FS, Onan U, Cox CF. A comparative scanning electron microscopic study on the characteristics of demineralized dentin root surface using different tetracycline HCl concentrations and application times. J Periodontol 2000;71:219-25.  Back to cited text no. 2
    
3.Wirthlin M.R. The current status of new attachment therapy. J. Periodontol, 1981;52:529-44.  Back to cited text no. 3
    
4.Berry EA 3 rd , von der Lehr WN, Herrin HK. Dentin surface treatments for the removal of the smear layer: An SEM study. J Am Dent Assoc 1987;115:65-7.  Back to cited text no. 4
    
5.Jones SJ, Lozdan J, Boyde A. Tooth surfaces treated in situ with periodontal instruments. Scanning electron microscopic studies. Br Dent J 1972;132:57-64.  Back to cited text no. 5
    
6.Register AA, Burdick FA. Accelerated reattachment with cementogenesis to dentin, demineralized in situ. I. Optimum range. J Periodontol 1975;46:646-55.  Back to cited text no. 6
    
7.Nanda Kumar, Cheru R. Topography of the instrumented periodontally involved root surface treated with citric acid, EDTA and sodium hypochlorite. A. SEM Study. ISP Bulletin; 1992.  Back to cited text no. 7
    
8.Bjorvatn K, Skaug N, Selvig KA. Tetracycline-impregnated enamel and dentin: Duration of antimicrobial capacity. Scand J Dent Res 1985;93:192-7.  Back to cited text no. 8
    
9.Hanes PJ, O'Brien Garnick JJ. A morphological comparison of radicular dentin following root planing and treatment with citric acid or tetracyclines HCl. J Clin Periodontol 1991:18:660-8.  Back to cited text no. 9
    
10.Labahn R, Fahrenbach WH, Clark SM, Lie T, Adams DF. Root dentin morphology after different modes of citric acid and tetracycline hydrochloride conditioning. J Periodontol 1992;63:303-9.  Back to cited text no. 10
    
11.Lafferty TA, Gher ME, Gray JL. Comparative SEM study on the effect of acid etching with tetracycline HCl or citric acid on instrumented periodontally-involved human root surfaces. J Periodontol 1993;64:689-93.  Back to cited text no. 11
    
12.Trombelli L, Scabbia A, Zangari F, Griselli A, Wikesjö UM, Calura G. Effect of tetracycline HCl on periodontally-affected human root surfaces. J Periodontol 1995;66:685-91.  Back to cited text no. 12
    
13.Ingman T, Sorsa T, Suomalainen K, Halinen S, Lindy O, Lauhio A, et al. Tetracycline inhibition and the cellular source of collagenase in gingival crevicular fluid in different periodontal diseases. A review article. J Periodontol 1993;64:82-8.  Back to cited text no. 13
    
14.Seymour RA, Heasman PA. Tetracyclines in the management of periodontal diseases. A review. J Clin Periodontol 1995;22:22-35.  Back to cited text no. 14
    
15.Baker PJ, Evans RT, Coburn RA, Genco RJ. Tetracycline and its derivatives strongly bind to and are released from the tooth surface in active form. J Periodontol 1983;54:580-5.  Back to cited text no. 15
    
16.Demirel K, Baer PN, McNamara TF. Topical application of doxycycline on periodontally involved root surfaces in vitro: Comparative analysis of substantivity on cementum and dentin. J Periodontol 1991;62:312-6.  Back to cited text no. 16
    
17.Anil S, Beena VT, Vijaya Kumar T. Current concepts of root surface treatment in periodontal therapy a review. Ind Soc Periodontal 1992.  Back to cited text no. 17
    
18.Lowenguth RA, Blieden TM. Periodontal regeneration: Root surface demineralization. Periodontol 2000 1993;1:54-68.  Back to cited text no. 18
    
19.Madison JG, Hopett SD. The effects of different tetracyclines on the dentin root surfaces of instrumented, periodontally involved human teeth. a comparative scanning electron microscopic study. J Periodontol 1997;68:739-45.  Back to cited text no. 19
    
20.Garrett JS, Crigger M, Egelberg J. Effects of citric acid on diseased root surfaces. J Periodontal Res 1978;13:155-63.  Back to cited text no. 20
    
21.Terranova VP, Franzetti LC, Hic S, DiFlorio RM, Lyall RM, Wikesjö UM, et al. A biochemical approach to periodontal regeneration: Tetracycline treatment of dentin promotes fibroblast adhesion and growth. J Periodontal Res 1986;21:330-7.  Back to cited text no. 21
    
22.Wikesjö UM, Baker PJ, Christersson LA, Genco RJ, Lyall RM, Hic S, et al. A biochemical approach to periodontal regeneration: Tetracycline treatment conditions dentin surfaces. J Periodontal Res 1986;21:322-9.  Back to cited text no. 22
    
23.Sterrett JD, Simmons J, Whitford G, Russell CM. Tetracycline demineralization of dentin: The effects of concentration and application time. J Clin Periodontol 1997;24:457-63.  Back to cited text no. 23
    
24.Register AA, Burdick FA. Accelerated reattachment with cementogenesis to dentin, demineralized in situ. II. Defect repair. J Periodontol 1976;47:497-505.  Back to cited text no. 24
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]


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