Vol. 23 No. 02 Apr-Jun 2014

^{Haroon Rashid1}                                BDS, MDSC  (UK)

The Confocal Laser Scanning Microscope (CLSM) is a type of microscope which utilizes its electronic system for image processing whilst using a laser as the source of light.  The optical image sections it obtains are of superior resolution. CLSM focuses on a single plane and removes the interference caused by the light arriving from the different optical fields across the thickness of the sample. As the images gathered using the microscope are digital images, higher magnifications for optical microscopy can be obtained using the software¹.

The images obtained using CLSM are of higher magnification as compared to the images obtained using the conventional optical microscopy. These images have volumetric and texture details and it is not possible to obtain such details with the conventional ones. The microscope also allows the study of the specimens using transmitted or reflected light meaning that those samples which are not transparent may also be analyzed. Since confocal microscope has the ability to create sharp optical sections, the data gathered from a series of optical sections at short and regular intervals along the optical axis are also used to create a true 3D reconstruction. This is made possible using software which combines the 2D images to create a 3D rendition ².

Confocal microscopy has been widely used in medical and dental research and also for the clinical treatment of various diseases. Along with its research applications in cancer and Alzheimer’s disease, the technique is also commonly used in ophthalmology^3,4, angiogenesis⁵, gynecology⁶, and gastro-intestinal systems⁷. CLSM is routinely used in dentistry to analyze surface roughness, analysis of dental erosion, measuring the actual profile, the numerical roughness parameters and in the studies of micro-tensile bond strength ^8,9,10,11. It has been widely used for studying the surface topography and formation of bio film on dental implants and dental hard tissues ^9,12. Once the data stack has been obtained in the CLSM a non-tactile, non-destructive measurement of the surface roughness is possible. It is an invaluable tool of measurement in dentistry studies. It is also important to mention that confocal microscope maintains the samples under humid conditions and thus avoids the artifacts that may take place with the use of SEM drying specimen preparation techniques. It offers improved relationship between the signal and the noise.

Confocal microscopy, like other devices has some limitations particularly the resolution and the pinhole size ^13,14. It has inherent resolution limitations due to diffraction and the airy disk used in CLSM can limit the maximum resolution which can be attained using the device. The pinhole size, once decreased in CLSM leads to reduced signal to noise ratio and thus more fluorescence from the specimen may be required.

CLSM is widely used in Europe and North America and is one of the most expensive types of microscope available in the market. The microscope is currently being employed for detailed microscopic examination of the cornea in some hospitals across Pakistan. National institute of biotechnology and genetic engineering uses CLSM for research purposes. It is a valuable tool in dental research particularly for accessing the surface roughness of dental biomaterials and the formation of biofilm on dental tissues. However, in Pakistan it is not yet employed in dental research partly because of its cost and partly because of lack of knowledge. Irrespective of financial constraints, it is important that this wonderful device is made available to the local researcher to claim its place in contemporary literature.

REFERENCES

1. Minsky, M. Memoir on inventing the confocal microscope. Scanning 1988;10:128-138.
2. Wilson, T.; Carlini, A.R. Three dimensional imaging in confocal imaging systems with finite-sized detectors. J. Microsc. 1988;141:51-66.
3. Jalbert I, Stapleton F, Papas E, et al. In vivo confocal microscopy of the human cornea. Br J Ophthalmol 2003;87:225-236.
4. Muller LJ, Marfurt CF, Kruse F, et al. Corneal nerves: structure, contents and function. Exp Eye Res 2003; 76:521-542.
5. McDonald DM, Choyke PL. Imaging of angiogenesis: from microscope to clinic. Nat Med 2003;9:713-725.
6. Drezek RA, Richards-Kortum R, Brewer MA, et al. Optical imaging of the cervix. Cancer 2003;98:20152027.
7. Vanden Berghe P, Bisschops R, Tack J. Imaging of neuronal activity in the gut. Curr Opin Pharmacol 2001;1:563-567.
8. Mazzitelli C, Ferrari M, Toledano M, Osorio E, Monticelli F, Osorio R. Surface roughness analysis of fiber post conditioning processes. J Dent Res 2008;87:186-190.
9. Hallgren C, Reimers H, Gold J, Wennerberg A. The importance of surface texture for bone integration of screw shaped implants: an in vivo study of implants patterned by photolithography. J Biomed Mater Res 2001;57: 485-496.
10. Pohl M, Stella J. Quantitative CLSM roughness study on early cavitation-erosion damage. Wear 2002;252:501511.
11. Mannocci F, Sherriff M, Ferrari M, Watson TF. Microtensile bond strength and confocal microscopy of dental adhesives bonded to root canal dentin. Am J Dent. 2001;14:200-4.
12. Dige I, Nilsson H, Kilian M, Nyvad B. In situ identification of streptococci and other bacteria in initial dental biofilm by confocal laser scanning microscopy and fluorescence in situ hybridization. Eur J Oral Sci. 2007;115:459-67.
13. Inoue S, Spring K.R. Microscope image formation. In Video Microscopy the Fundamentals; Plenum Press: New York, 1997;13-118.
14. Wilson, T. The role of the pinhole in confocal imaging system. In Handbook of Biological Confocal Microscopy, 2nd Ed.; Pawley, J.B., Ed.; Plenum Press: New York, 1995;167-182.

1. Assistant Professor, Department of Prosthodontics, Ziauddin College of Dentistry, Ziauddin University, Karachi, Pakistan.

Corresponding author: “Dr. Haroon Rashid” < drh.rashid@hotmail.com >

Tel: : +92-323-3241889

Asma Naz¹                                               BDS, FCPS

Shujah Adil Khan²                               BDS

INTRODUCTION

Prosthesis replacing anterior teeth frequently look artificial because teeth which have been selected are smaller than the natural teeth which they are replacing¹, and this is considered a problem in fabricating dentures. With the absence of pre-extraction records³, the selection of appropriate size of maxillary anterior teeth is one of the most confusing and difficult aspect of complete denture construction³ Suitable positioning has always posed great challenges⁴. The best position for placing artificial teeth is the one occupied by its predecessors⁵. When pre-extraction records are available the task is simple⁶.  Several methods have been employed for placement in positions of natural predecessors⁷.

Multiple landmarks have been used and the most common being the incisive papilla⁴, labial vestibule⁵, retromolar pad⁸, palatal vestigial remnants⁹, midpalatal suture¹⁰ and palatal rugae.

The incisive papilla (IP) is a stable and noticeable anatomical landmark which survives during the progression from dentate to edentulous⁴. It is an important landmark as it is a stable structure and usually does not shift in adult life^11,12. It serves to provide a guide to determine the midline¹⁰, the labio palatal position of prosthethic incisors and canines, inciso-cervical position of incisors⁴, a starter for the occlusal rim fabrication and the central incisor region¹³ and determination of parallelism of the occlusal plane, when used in conjunction with hamular notch¹⁴.

The distance of IP from the central incisors has been measured from the center⁶, or posterior border of the papilla^4,5,8,9,15. This distance also varies in the different arch forms. Studies have been conducted for other populations such as Caucasian^4,5,10, Chinese⁹, Thai⁸, Korean¹⁵ and Taiwanese populations and Pakistani populations as well.

The present study aims at documenting this distance in dental students of two classes in a dental hospital of Karachi, Pakistan with different arch forms. This will reduce the dentist’s chair side effort and patient’s time by allowing the dental laboratory technicians to reproduce the relationship established between the natural teeth and the supporting facial structures⁵, especially in the absence of pre extraction records.

METHODOLOGY

A cross sectional in-vitro experimental study was carried out over a period of 6 months from March 2012 t o August 2012 at Department of Prosthodontics, Dr Ishrat ul Ebad Khan Institute of Oral Health Sciences, DUHS. Undergraduate students of 3^rd and 4^th years were selected for sampling, and all of the students ranging in age between 20 and 22 years. The selection criteria were based on healthy dentitions, well aligned arches with all incisors, canines, first and second premolars and molars present. Subjects with malposed teeth, periodontal disease, restored anterior teeth, history of orthodontic treatment, congenital and/or acquired maxillary defects, orthognathic/reconstructive surgical procedures were excluded.

Maxillary impressions were made with Irreversible hydrocolloid (Tropicalgin by Zhermack Spa) following the manufacturer instructions for mixing using the supplied water powder measuring scoop and cylinder. It was hand mixed in a rubber bowl and loaded onto stainless steel perforated stock trays. After introduction of the tray into the patient’s mouth the material was allowed to set for two and a half minutes to ensure an adequate final set. The impression was removed, inspected, washed and disinfected for ten minutes and casts were made using type 4 dental stone and bases were made using a standard base former.

The casts were placed on a horizontal surface and incisive papilla was first identified and then boundaries marked with lead pencil. Distance from posterior surface of IP to maximum convexity of central incisor, the papillo incisal distance (PID) was measured with a modified vernier caliper:  The fixed jaw blade of the vernier caliper extended 10 mm from the original for effective measurements. The arch forms were assessed by their basic morphological description classified as Ovoid, Tapering and Squarish.

The collected data was entered, and analyzed using SPSS program version 16. The study variables included “gender”, “PID” and “Arch Form”. Comparative mean were calculated for the variables defined in the study.

RESULTS

Among 203 subjects about 90% were females as being dominant in the institution and the age group range in between 20 – 22 years. The mean PID was 11.06 mm ± SD 1.46. Among the subjects were 90.6% had ovoid, 3.9% had squarish and 5.4% had tapering arch forms. Mean PID for Ovoid arches were found to be 11.04mm, Tapering 10.84mm and Squarish 12.02mm.

DISCUSSION

Arranging artificial teeth according to the anatomical landmarks helps in positioning the teeth in relation to the general arch form and one another. The pre-maxilla may be referred to as an “esthetic zone” because of its high visibility and influence on facial appearance. Improper positioning of maxillary centrals may result in distorted appearance and may affect speech¹⁶.

Many geometric arch forms and mathematical functions have been proposed to describe dental arches over the years^17,18. Some authors^5,19,20 prefer to classify dental arches into ovoid, tapering and squarish, this classification of arch forms was used in the present study. The majority of the subjects had an ovoid arch form 90% where as 6% had tapering and 4% had squarish arch forms. In a comparison of Japanese and Caucasian mandibular dental arches¹⁵ the Japanese group had 46% squarish arches, 42% ovoid and 12% tapering arches. The Caucasian group had 18% squarish arches, 38% ovoid and 44% tapering. The author reported that there were ethnic differences in arch forms. This explanation may also be relevant to the Pakistani samples as reported

Papillo-Incisal Distance

by Zia²¹. PID is an important biometric guide for positioning of maxillary occlusal rim and central

Statistics

incisors²². Mean PID measured in this study sample was 11.06mm SD±1.46. In Grave’s¹ study with the same landmarks the mean PID was 13.1mm on dentate subjects in Caucasians. The mean distance was 12.45mm in another Caucasian sample²³. In a Thai sample it was

Frequencies

11.093mm and 12.269mm from incisive papilla to incisal edge of the central incisor and the most convex labial surface respectively⁸. In a study on Southern Chinese population the posterior limit as well as center of the incisive papilla. to the most labial contour of the central incisor was used⁹. Mean value for the distances were 12.71mm and 9.17mm respectively. A Jordanian study recorded 12.93mm from incisal tangent to the posterior limit of the incisive papilla using computer scanner and software program²⁴. In a 3D orthographic study on Korean sample the PID from the posterior border of the incisive papilla to a vector drawn in the midpoint of mesio-incisal tips of both maxillary incisors was 11.96mm¹⁵. The PID in studies using posterior limit of the incisive papilla as the reference point for measurement is comparable to this study.

The present study was designed to discover various arch forms in the collected sample and to find relation of PID in these arch forms. The ovoid arches were dominant i.e. 82.4%, the other two arch forms combined were only 17.8% which may have affected the final outcome. This could be a limitation of present study and use of equal number of the three arch forms may be explored in future research projects.

CONCLUSIONS

The suggested mean PID for placing prosthetic central incisors is 11.06mm. There is a higher presentation of ovoid arch forms.

REFERENCES

1. Kassab NH. The selection of maxillary anterior teeth width in relation to facial measurements at different types of face form. Al- Rafidain Dent J. 2005; 5:15-23.
2. Hoffman W, Bomberg TJ, Hatch RA. Interalar width as a guide in denture tooth selection. J Prosthet Dent. 1986;55:219-221.
3. Woodhead CM. The mesiodistal diameter of permanent maxillary central incisor teeth and their prosthetic replacement. J Dent. 1977;5:93-98.
4. Grave AMH. Evaluation of incisive papilla as a guide to anterior tooth position. J Prosthet Dent. 1987;5: 712-714.
5. Mersel A, Ehrlich J. Connection between incisive papilla, central incisor and rugae canina. Quintessence Int. 1981; 12: 1327-1329.
6. Saleem T, Ahmad TZ. Incisive papilla maxillary incisor distance in subjects of various arch forms. Professional Med J 2011;18:644-648.
7. Rufenacht CR. Structural esthetic rules. In: Rufenacht CR, editor. Fundamental of esthetics. Chicago: Quintessence Publishing Co Inc; 1990:p.67134.
8. Chatsuthipan S, Boonsiri I, Wongthai P. Relationship of central incisor and canine to incisive papilla. CU Dent J. 1993;16:29-40.
9. Lau GCK. The relationship of incisive papilla to the maxillary central incisors and canines in southern Chinese. J Prosthet Dent. 1993;70:86-93.
10. Roraff AR. Arranging artificial teeth according toanatomic landmarks. J Prosthet Dent. 1977;38:120-30.
11. Ehrlich J, Gazit E. Relationship of the maxillary central incisors and canines to the incisive papilla. J Oral Rehab 1975;2:309-12.
12. Huang SJ, Chou TM, Lee HE, Wu YC, Yang YH,Ho CD, et al. Exploring the distance between upper central incisor edge and incisive papilla in Taiwanese population. Taiwan J Oral Med Health Sci 2004; 20:4-10
13. Guldag MU, Sentut F, Buyukkplan US. Investigationof vertical distance between incisive papilla and incisal edge of maxillary central incisors. Eu J Dent:2008;2: 161-166.
14. Fu PS, Hung CC, Hong JM, Want JC. Threedimensionalanalysis of occlusal plane related to the hamular-incisive-papilla occlusal plane in young adults. J oral rehab. 2007;34:136-140.
15. Park YS, Lee SP, Paik KS. The three-dimensionaland relationship on a virtual model between the maxillary anterior teeth and incisive papilla. J Prosthet Dent 2007; 98:312-318.
16. Runte C, Lawerino M, Dirkson D, Bollmann F,Lamprechi- Dinnesen A, Seifert E. The influence of maxillary central incisor position in complete denture on /s/ sound production. J Prosthet Dent. 2001;85:485495.
17. Noroozi H, Nik HT, Saeeda R. The dental arch formrevisited. Angle Orthod. 2001;71:386-389.
18. Broomell, editor. In: Anatomy and histology of themouth teeth. 2nd ed. Philadelphia: P. Blakiston’s Son & Co 1902:99.
19. Kunihiko N, McLaughlin RP, Isshiki Y, SinclairPM. A comparative study of Caucasian and Japanese mandibular clinical arch forms. Angle Orthod. 2001; 71:195-200.
20. Chuck GC. Ideal arch form. Angle Orthod. 1934;4: 312-327.
21. Zia M, Azad AA, Ahmed S. Comparison of distancebetween maxillary central incisors and incisive pailla in dentate individuals with different arch forms. J Ayub Med Coll Abbottabad. 2009;21:125-128.
22. Kamashita Y,  Kamada  Y,  Kawahata  N  andNagaoka E. Influence of lip support on the soft tissue profile of complete denture wearers. J Oral Rehabil. 2006;33:102-109.
23. Ortman HR, Tsao DH. Relationship of the incisivepapilla to the maxillary central incisors. J Prosthet Dent. 1979;42:492-496.
24. Amin WM, Taha ST, Al-Tarawneh SK, Saleh M,Ghzawi A. The Relationships of the Maxillary Central Incisors and Canines to the Incisive Papilla in Jordanians. J Contemp Dent Pract. 2008;5:42-45.

1. Assistant Professor and Head of department, Department of Prosthodontics, Dr. Ishrat ul Ebad Khan Institute of Oral Health Sciences, Dow University of Health Sciences, Karachi.
2. MDS Resident Department of Prosthodontics, Dr. Ishrat ul Ebad Khan Institute of Oral Health Sciences, DUHS, KarachiCorresponding author: “Dr. Shujah Adil Khan” < shujah.adil@gmail.com >

Fahim Ahmed Vohra¹                             BDS, MFDS, MClinDent, MRD RCS

Syed Rashid Habib²                                BDS, FCPS

INTRODUCTION

The fabrication process of an indirect restoration includes multiple clinical and laboratory steps, each of which have the potential to introduce inaccuracies in the definitive restoration.  If a dental prosthesis is to be an accurate fit, it needs to be made on an accurate master cast (1-3). According to ISO (International standards organization) die material must comply with certain standards (4), some of which are good dimensional accuracy, accurate detail reproduction, durability and abrasion resistant, thixotropic behavior, ease of manipulation, command set, compatibility with other materials and bio-compatibility (4-7). In conventional prosthodontics a number of materials have been suggested for master cast fabrication.  These include die stone, resin reinforced die stone, resins and metal plated dies. Out of those materials that fulfill the requirements of American Dental Association (ADA), type IV dental stone (high strength, low expansion) is almost universally used in dentistry for fabrication of dies and cast in the process of prosthesis fabrication (8).

Reason being, their favorable handling properties, low cost and predictable production of slightly oversized dies when used with elastomeric impression materials. Unfortunately, die stones have poor mechanical properties with low fracture strength and abrasion resistance (6). This means that they can be very easily damaged during complex laboratory procedures.  Indeed, it is for these reasons that alternative die materials have been investigated.  In order to improve the performance of die stone, resin-reinforced die stones have been developed. However, their surface detail, dimensional stability and hardness were similar to conventional type IV dies stones (6). Copper and silver plated dies have demonstrated exceptional mechanical properties along with good detail reproduction and dimensional stability (9,10). Their routine use may however be precluded by the fact that they are expensive, time consuming to produce and their production is technique sensitive requiring specialist equipment (10).  In addition, silver plating is accomplished using a silver cyanide solution, which may produce toxic cyanide gas if mixed with acid.  However, in more complex cases involving multiple pick-up impressions their use may be preferred due to superior durability.

Resin die materials are available in the form of epoxy and polyurethane resin. Duke et al,(6) compared transverse strength of different die materials, by three point testing to failure. Die stones had three times less strength as compared to resins. Abrasion resistance of resin dies are shown to be second to metal dies but is greater in comparison to conventional and modified die

stones (6). Resins also demonstrated accurate detail reproduction, recording a line of upto 1 micrometer, whereas dental stone and resin-reinforced stones could only record as low as 17 micrometer (6). Initial reports for dimensional changes of resin dies were unfavorable, revealing shrinkage of 0.09% (6). A recent investigation of contraction levels for epoxy resin using different polymerization methods, reported significant contraction variability, deeming them inaccurate for master cast fabrication (11).  Epoxy resin also requires an electric centrifuge to decrease porosity and increase accuracy of master cast. Interestingly, shrinkage for polyurethane resin materials can be controlled by varying silica filler proportions. A polyurethane resin with 60% filler was shown to have transverse strength, which is 2.5 times of dental stone superior to epoxy resin (12). In clinical cases with tall and thin tooth preparations, removal of master cast from elastic impressions most likely result in fractures. Adequately filled polyurethane resin materials, which along with ease of manipulation have increased transverse strength, can perform as an ideal master cast/ die material.

This report will present a clinical case demanding the use of polyurethane resin die material for increased fracture resistance and accurate detail reproduction of multiple tall and thin tooth preparations, in a generalized tooth wear patient.

CASE REPORT

A 57-year-old male patient presented to Edinburgh Dental Institute, Restorative department, with severe maxillary tooth wear and missing maxillary teeth. After complete assessment, which included history, examination, special investigation, articulation and waxup of study casts, the patient was diagnosed to have severe tooth wear in maxilla, moderate tooth wear in mandible (erosion), generalized moderate to severe bone loss in maxilla and mild to moderate bone loss in mandible (from treated periodontal disease) (Fig 1), lack of posterior occlusal stability and missing maxillary right posterior teeth (Fig 2). The definitive treatment plan was executed in the following treatment sequence; (1) Placement of implants at 14 & 16, (2) Gold shell crown (GSC) preparation of teeth 36 & 46 (3) Leveling of the mandibular occlusal plane, (4) Crown preparation

of teeth 13 to 25 (5) Extraction of tooth 24, (6) Provisionalization of complete dentition at the raised OVD (7) Replacement of temporary contact stops with composite, (8) Provision of PFM crowns 13-22, (9) Provision of PFM bridge 23-25, (10) Provision of implant retained PFM bridge 14-16,  (11) Provision of gold shell crowns 36  & 46.

After steps 1 to 7 were successfully completed, individual impressions of teeth were recorded after soft tissue displacement using retraction cords impregnated ( Ultrapak, Ultradent Inc, St Louis, USA) with astringent ( Viscostat , Ultradent Inc, St Louis, USA ). Impressions were recorded using dual phase heavy-light technique (Express VPS, 3M ESPE, USA) in PVS material in a stock tray. A decision was made to fabricate individual dies using polyurethane material (AlphaDie MF, Schutz Dental Group, Sylmar, CA,USA) and verify their accuracy by transfer copings. Equal amounts of resin base and filler were mixed in a plastic pot with a wooden spatula and die hardener was added according to

manufacturers instructions to the mix.  Pouring of the impression was done on a vibrator within five minutes and complete set was achieved in three to five hours. Individual dies were trimmed and resin (Duralay inlay pattern resin, Reliance Dental Mfg corp, Worth Il, USA) pick copings were fabricated on each die (fig 3 & 4). A

pick up master impression was recorded after splinting the individually seated transfer impression copings in PVS material using a dual phase heavy light consistency technique (fig 5). Registration in RAP was recorded using wax (Alminax Rite Bite, Swindon, UK) and ZOE (TempBond, Kerr, CA,USA). Crowns (13-22) and bridge (23-25) were cemented using a resin based luting cement to enhance retention of the restorations.

Cement retained implant fixed partial denture (14- 16) and GSC’s were cemented to complete the case. The

patient was pleased with the outcome (fig 6) and was reviewed at 3, 6, 12 months, followed by a yearly review for maintenance.

DISCUSSION

Patient was delighted with the outcome of the treatment of his severely worn teeth and was pleased with the fixed replacement of the missing maxillary teeth.  Attention to detail at each stage was crucial to the treatment provision and the successful outcome. Patient expectations were identified in the beginning and considered in all treatment decisions¹³. A diagnostic wax up was completed at the start of the treatment and a mock up in the mouth was performed to allow the patient to assess the proposed treatment results. A reorganized approach was used for rehabilitation of the patient, as space lost due to tooth wear was gained by increasing the VDO^14,15.

The prepared maxillary teeth had moderate to severe bone loss from previous periodontal disease¹⁶. Firstly, this rendered preparations to be tall and narrow as margins had to be placed equi-gingival for the purpose of aesthetic and maintenance. Preparations were kept conservative, using putty indices from the diagnostic wax up¹⁷ and immediate dentine sealing was done¹⁸. In order to avoid fracture of dies, resin instead of die stone was used¹². Secondly, periodontal tissues were highly susceptible to breakdown due to further disease and trauma from

prosthodontic procedures; hence a complete mouth pickup impression using splinted customized copings was used¹⁹. This allowed for minimal trauma as prolong and repeated soft tissue retraction was avoided and aided prosthesis fabrication as soft tissues were recorded in rest state. Lastly, possible tooth mobility of the periodontally involved teeth was prevented by splinting resin copings in the pick-up master impression. Impressions were verified by making individual resin dies, these were in turn verified in the mouth-using pick up copings prior to impression making.

The choice for die material was between metal platted and resin dies, as resin reinforced stone dies have shown mechanical properties almost similar to type IV stone dies⁶. Metal plated dies are dimensionally accurate, have smooth surfaces and are durable with high strength and abrasion resistance^9,20. However unavailability of special electroplating bath and biological hazard of the materials involved prevented their use. Resins provide excellent surface detail, good abrasion resistance, adequate transverse strength (2.5 times of type IV stone)¹². In contrast to epoxy resin, impressions can be accurately poured without requiring a centrifuge with polyurethane resin. The suggested shrinkage for resin (Alpha Die MF) by the manufacturer is 0.06% (low shrinkage), this change however was compensated by increasing the filler particles of the mix to 60%¹². Handling of the polyurethane resin was challenging, as the properties of the material are highly sensitive to manipulation technique. The material is tacky and sticks to instruments (disposable instruments were used) and has low thixotropy as a result pouring was carried out on maximum vibrations to avoid porosities.

All efforts were made to maintain healthy soft tissue during the provisional phase, which provided an opportunity for assessment of aesthetics and occlusion. Patient was informed that maintenance of good plaque control and excellent OH was essential for the good prognosis of the treatment.

CONCLUSION

In summary, this case report displays the use of polyurethane resin as a viable alternative to die stone for master cast and die fabrication in complex prosthodontic cases, for enhancing the quality of definitive restorations and ensuring successful clinical outcome.

REFERENCES

1. Vigolo P, Millstein PL:Evaluation of master cast techniques for multiple abutment implant prostheses. Int J Oral Maxillofac Implants 1993; 8:439-446
2. Dilts WE, Podshadley AG, Sawyer HF, et al: Accuracy of four removable die techniques. J Prosthet Dent 1971; 83:1081-1085
3. MyersM, Hembree JHJr :Relative accuracy of four removable die systems. J Prosthet Dent 1982; 48:163165
4. Anusavice KJ: Phillip’s Science of Dental Materials (ed10). Philadelphia, PA, Saunders, 1996, p. 186
5. Brabant A, Grimonster J, Deschamps R,VanBiervlietL. Comparative study of four physical properties of 13 dental stones, “hard” and “extra hard” [in French] Cah Prothbse 1980; 32:145-157.
6. Duke P, Moore BK, Haug SP, Andres CJ. Study of the physical properties of type IV gypsum, resincontaining, and epoxy die materials. J Prosthet Dent 2000 ;83: 466- 473.
7. Bailey JH, Donovan TE, Preston JD. The dimensional accuracy of im- proved dental stone, silverplated, and epoxy resin die materials. J Prosthet Dent 1988;59:307310.
8. AmericanNationalStandardsInstitute/AmericanDental Association: Specification No. 25 for Dental Gypsum products, 2000
9. Toreskog S, Phillips RW, Schnell RJ. Properties of die materials: A comparative study. J. Prosthet Dent. 1966;16:119-131.
10. Cassimaty EM, Walton TR: Effect of three variables on the accuracy and variability of electroplated copper dies. Int J Prosthodont 1996;9:547-554.
11. Prisco R, Cozzolino G, Vigolo P. Dimensional accuracy of an epoxy die material using different polymerization methods. J Prosthodont. 2009;18:156-161.
12. Derrien G, Sturtz G. Comparison of transverse strength and dimensional variations between die stone, die epoxy resin, and die polyurethane resin. J Prosthet Dent. 1995;74:569-574.
13. Ibbetson R. Tooth surface loss. 9. Treatment planning. Br Dent J. 1999; 186:552-558.
14. Dyer K, Ibbetson R, Grey N. A question of space: options for the restorative management of worn teeth. Dent Update 2001; 28:118-123.
15. Davies SJ, Gray RM, Whitehead SA. Good occlusalpractice in advanced restorative dentistry. Br Dent J. 2001;191:421-434.
16. Nyman S, Lindhe J. A longitudinal study of combinedperiodontal and prosthetic treatment of patients with advanced periodontal disease. J Periodontol. 1979; 50:163-169.
17. Magne P, Belser UC. Novel porcelain laminate preparation approach driven by a diagnostic mock-up. J Esthet Restor Dent. 2004; 16:7-16.
18. Magne P. Immediate dentin sealing: a fundamentalprocedure for indirect bonded restorations. J Esthet Restor Dent. 2005;17:144-154
19. Wallace FH. Resin transfer copings. J Prosthet Dent1957;8:289-292
20. Fan PL, Powers JM, Reid BC. Surface mechanicalproperties of stone, resin, and metal dies. J Am Dent Assoc. 1981;103:408-411

1. Assistant Professor, Prosthetic Dental Science Department. Address: SDS, College Of Dentistry, King Saud University
2. Assistant Professor, Prosthetic Dental Science Department.College Of Dentistry, King Saud University.

Corresponding author: “Dr. Fahim Ahmed Vohra” < fahimvohra@yahoo.com >

Azizah F. Bin Mubayrik¹                         BDS, MSc, Clin Cert

K. Deeley²                                                    MPH

A. Patir³                                                       DDS, PhD

M. Koruyucu⁴                                            DDS, PhD

F. Seymen⁵                                                 DDS, PhD

A.R. Vieira⁶                                                DDS, MS, PhD

INTRODUCTION

Dental caries is a model multifactorial disease.

While the cariogenic microorganism Streptococcus mutans plays a dominant role in the pathogenesis of dental caries, the development of the disease is also intimately linked to both substrate (amount of fermentable carbohydrate, protective foods, pH) and host (tooth anatomy, enamel quality, immune competence, and saliva quantity and quality) factors [1]. As a component of host-related factors, the importance of genetic susceptibility has been recognised for decades, with caries being attributable to an estimated 30-60% heritable component [2]. However, although a few candidate genes (such as tuftelin [3] and ameloblastin [4] are postulated to contribute to the pathogenesis via plausible mechanisms, these associations have not been rigorously replicated and there are no useful genetic biomarkers for use in practice. The results of genomewide association studies (GWAS) may provide an unbiased appraisal of susceptibility loci, and these have started to report in both adults [5] and children [6]. However, the results for children in particular have been disappointing, with no loci meeting criteria for genomewide significance.

However, a recent study measuring three single nucleotide polymorphisms (SNPs) in the promoter region of the β-defensin gene DEFB1 demonstrated a significant association with dental caries in the permanent dentition of nearly three hundred unrelated individuals [7]. The variant allele rs11362 conferred a five-fold increased risk in both decayed, missing teeth due to caries, filled teeth (DMFT) and decayed, missing teeth due to caries, filled surface of tooth (DMFS) scores, while the variant rs119946 genotype was associated with a reduced risk. The mechanistic hypothesis is plausible: defensins are key mediators of innate immunity and are a first line defence against pathogens such as S. mutans, and therefore any genetic variability that alters the efficacy of defensins may also influence microbial colonisation or pathogenicity [8]. In addition, DEFB1 has been shown to be constitutively expressed in oral cells and tissues when oral micro-organisms are present [9-11], and DEFB1 is secreted in crevicular fluid [12] and saliva [13], suggesting importance in oral health.

These promising results in adults prompted us to investigate the association between two SNPs in DEFB1, (rs11362 and rs1800972) and dental caries in the primary dentition of children aged three to six years of age. We aimed to establish whether DEFB1 polymorphisms contribute to early disease pathogenesis and therefore what clinical role, if any, genetic testing of DEFB1 might play in early detection and active management of dental caries in children.

SUBJECTS AND METHODS

Study subjects and definitions

A total of 178 children aged between three and six years of age were recruited into the study at the Department of Pedodontics, Istanbul University. All parents provided written informed consent for the participation of their children in the study and this protocol was approved by University of Pittsburgh Institutional Review Boards (PRO11070236).

Individuals were categorised as either controls (no caries; n=86) or as having dental caries (n=92); dental caries was defined as individuals with either a DMFT or DMFS of >1. For more stringent analysis of the caries phenotype, ‘high caries’ was defined as DMFT of >8, or DMFS equal to or greater than their age, as described previously [7].

Genotyping

Unstimulated saliva samples were obtained from all participants and stored in Otagene DNA self-collection kits (DNA Genotek Inc., Ottawa, ON, Canada) at room temperature until processed. DNA was extracted according to the manufacturer’s instructions.

Two polymorphisms (rs11362 (G-20A) and rs1800972 (C-44G)) in the promoter region of DEFB1 were selected for study, as previously described [Ozturk et al., 2010]. The variants were genotyped using a validated Taqman assay on an ABI Prism 7900HT Sequence Detection System and automated software (Applied Biosystems, Foster City, CA).

Statistical analysis

Goodness of fit to Hardy-Weinberg expected proportions was examined using the chi-squared test. Pairwise linkage disequilibrium was estimated with D’ [14]. Differences between groups were tested using the Student’s t-test or chi-square test depending on whether the data were continuous or categorical, respectively. Odds ratios were determined using the 2-way contingency table chi-square test. Odds ratios were calculated by comparing individuals with zero copies of the variant allele and individuals with 1 or 2 copies of the variant allele. Statistical analysis was performed using SPSS® for Windows (IBM® SPSS® Statistics 19, Chicago, III, USA), and a p-value of <0.05 was considered statistically significant.

RESULTS

The mean age of the participants was 5.37 years, and ranged from three to six years of age. Average age of children in the caries group was 4.83 years old. Both groups were equally matched for gender (Table 1). Children with dental caries had DMFT scores ranging from 1 to 15 (average 7.0) and DMFS scores ranging from 1 to 34 (average 9.9).

Legends

Table 1. Demographics data of the Sample according to caries status.

The rs11362 and rs1800972 polymorphisms were in linkage disequilibrium (p=xxx) and the genotypes in Hardy-Weinberg equilibrium. Allele frequencies were similar to previously described [7]. There was no evidence of an association between polymorphisms in DEFB1 and the presence or absence of dental caries (Table 2), with the odds ratios for rs11362 (G-20A) and rs1800972

Table 2. Distribution of DEFB1 Promoter Polymorphisms Based on Caries Experience.

(C-44G) 0.93 (0.51-1.69) and 1.08 (0.55-2.01), respectively. Even when considering the extremes of caries phenotype, there was no association between genotype and phenotype.

DISCUSSION

Polymorphisms in the promoter region of DEFB1 have recently been shown to be associated with the caries experience in the permanent dentition of adults [7]. This prompted us to investigate whether DEFB1 SNPs were also associated with caries in primary dentition, in order to provide insights into the role that DEFB1 might play in the pathogenesis of dental caries and establish in which patient groups DEFB1 SNP testing might be clinically useful. We did not find an association between the two SNPs tested and caries in the primary dentition in this cohort of unrelated children, suggesting that either DEFB1 does not influence disease progression in children, other factors predominate in the pathogenesis of dental caries in children, or that DEFB1 mediates long-term or chronic disease progression. For instance, in addition to their role in innate immunity, defensins also mediate the cross-talk between innate and acquired immune responses, such as by acting as chemoattractants for T-cells and dendritic cells [15]. Their effects may therefore not be evident over the relatively short timeframes represented in the genesis of caries in children.

The results of GWAS studies into dental caries have largely been disappointing, especially in children. In a large GWAS study of 1305 children aged 3-12 years old, there were no single SNPs that were definitively associated with childhood dental caries, although a few regions on chromosomes 1, 11, and 17 did highlight weak associations with caries and contained a few plausible candidate genes of interest, including ACTN2,

MTR, EDARADD, MPPED2, and LPO [6]. Weak associations with caries have also been observed for regions near the DEFB1 locus at 8p23.1 in another small GWAS study, although the locus itself was not significant [16]. Adult studies have been slightly more encouraging, with two homologous genes (BCOR and BCORL1) found to be associated with the phenotype [5]. Although the heritability of caries scores in primary dentition has been reported to be greater than in permanent dentition [17], this observation does not appear to be borne out when tested by modern genotyping methodologies that have proven to be so successful for determining susceptibility to other complex genetic traits, such as for cardiovascular disease. Together with the current study, however, it appears that the bacterial, dietary, salivary, morphological, hygienic, and exposure-related factors may dominate in children.

This study is not without limitations. The sample size is relatively small and data on diet, oral hygiene, bacterial colonisation, and fluoride exposure, as well as other significant demographic details such as ethnicity and socioeconomic status are not available. These factors are known to influence risk and might provide a greater insight into the influencing factors in this cohort. Since gene-environment interactions are likely to be important in the pathogenesis of caries, the genetic risk is likely to be environment-specific, and further attempts to establish genetic risk stratifiers will need to take this into account.

In conclusion, although polymorphisms in the promoter region of DEFB1 appear to be associated with caries risk in adults, they do not appear to be associated with risk of caries in primary dentition in children, consistent with genome-wide studies. Other non-genetic factors appear to be greater modifiers of risk and are likely to be more useful for stratification purposes.

REFERENCES

1. Shulman JD, Cappelli DP: Chapter 1 – epidemiology of dental caries; in: Prevention in clinical oral health care. Saint Louis, Mosby.2008: pp 2-13.
2. Boraas JC, Messer LB, Till MJ: A genetic contribution to dental caries, occlusion, and morphology as demonstrated by twins reared apart. J Dent Res. 1988;67:1150-1155.
3. Slayton RL, Cooper ME, Marazita ML: Tuftelin, mutans streptococci, and dental caries susceptibility. J Dent Res. 2005;84:711-714.
4. Deeley K, Letra A, Rose EK, Brandon CA, Resick JM, Marazita ML, Vieira AR. Possible association of amelogenin to high caries experience in a guatemalanmayan population. Caries Res. 2008;42:8-13.
5. Zeng Z, Shaffer JR, Wang X, Feingold E, Weeks DE, Lee M, Cuenco KT, Wendell SK, Weyant RJ, Crout R, McNeil DW, Marazita ML: Genome-wide association studies of pit-and-fissure- and smooth-surface caries in permanent dentition. J Dent Res. 2013;92:432-437.
6. Shaffer JR, Wang X, Feingold E, Lee M, Begum F, Weeks DE, Cuenco KT, Barmada MM, Wendell SK, Crosslin DR, Laurie CC, Doheny KF, Pugh EW, Zhang Q, Feenstra B, Geller F, Boyd HA, Zhang H, Melbye M, Murray JC, Weyant RJ, Crout R, McNeil DW, Levy SM, Slayton RL, Willing MC, Broffitt B, Vieira AR, Marazita ML: Genome-wide association scan for childhood caries implicates novel genes. J Dent Res. 2011;90:1457-1462.
7. Ozturk A, Famili P, Vieira AR: The antimicrobial peptide defb1 is associated with caries. J Dent Res. 2010;89:631-636.
8. Weinberg A, Krisanaprakornkit S, Dale BA: Epithelial antimicrobial peptides: Review and significance for oral applications. Crit Rev Oral Biol Med, 1998;9:399-414.
9. Dunsche A, Acil Y, Siebert R, Harder J, Schroder JM, Jepsen S: Expression profile of human defensins and antimicrobial proteins in oral tissues. J Oral Pathol Med.  2001;30:154-158.
10. Krisanaprakornkit S, Weinberg A, Perez CN, Dale BA: Expression of the peptide antibiotic human betadefensin 1 in cultured gingival epithelial cells and gingival tissue.  Infect Immun. 1998;66:4222-4228.
11. Mathews M, Jia HP, Guthmiller JM, Losh G, Graham S, Johnson GK, Tack BF, McCray PB, Jr.: Production of beta-defensin antimicrobial peptides by the oral mucosa and salivary glands. Infect Immun. 1999 Jun; 67:2740-2745.
12. Diamond DL, Kimball JR, Krisanaprakornkit S, Ganz T, Dale BA: Detection of beta-defensins secreted by human oral epithelial cells. J Immunol Methods. 2001;256:65-76.
13. Sahasrabudhe KS, Kimball JR, Morton TH, Weinberg A, Dale BA: Expression of the antimicrobial peptide, human beta-defensin 1, in duct cells of minor salivary glands and detection in saliva. J Dent Res. 2000;79:16691674.
14. Lewontin RC: The interaction of selection and linkage. I. General considerations; heterotic models. Genetics. 1964;49:49-67.
15. Yang D, Chertov O, Bykovskaia SN, Chen Q, Buffo MJ, Shogan J, Anderson M, Schroder JM, Wang JM, Howard OM, Oppenheim JJ: Beta-defensins: Linking innate and adaptive immunity through dendritic and T cell ccr6. Science. 1999;286:525-528.
16. Vieira AR, Marazita ML, Goldstein-McHenry T: Genome-wide scan finds suggestive caries loci. J Dent Res. 2008;87:435-439.
17. Wang X, Shaffer JR, Weyant RJ, Cuenco KT, DeSensi RS, Crout R, McNeil DW, Marazita ML: Genes and their effects on dental caries may differ between primary and permanent dentitions. Caries Res. 2010;44:277-284.

1. Dept. of  Oral Medicine & Diagnostic Sciences College of Dentistry, King Saud University, Riyadh; Saudi Arabia.
2. Research Specialist, Department of Oral Biology, School of Dental Medicine, University of Pittsburgh, Pittsburgh, PA, USA
3. Assistant Professor, Department of Pedodontics, Faculty of Dentistry, Medipol Istanbul University, Istanbul, Turkey
4. Assistant Professor, Department of Pedodontics, Faculty of Dentistry,  Istanbul University, Istanbul, Turkey
5. Professor, Department of Pedodontics, Faculty of Dentistry, Istanbul University, Istanbul, Turkey
6. Associate Professor, Departments of Oral Biology and Pediatric Dentistry, Center for Craniofacial and Dental Genetics, and Clinical and Translational Science Institute, University of Pittsburgh, Pittsburgh, PA, USA

Corresponding author: “Azizah F. Bin Mubayrik ” < azizafm2001@yahoo.com >

Aiyesha Wahaj¹                         BDS

Imtiaz Ahmed²                         BDS, FCPS, M.ORTH

Gul-e-Erum³                              BDS, FCPS, M.ORTH

INTRODUCTION

Craniofacial anomalies include a diverse group of deformities related to development of the head and neck. Their etiology shows complexity like apert syndrome, crouzon syndrome etc. They all are either entirely genetic in origin while some are due to teratogenic influences or may be associated with any other developmental anomalies including cleft lip and palate. Their incordinated growth behavior evokes craniofacial characteristics which are significantly deviated from normal and most commonly presented as craniofacial asymmetry.

Asymmetry refers to differences in size, shape or relative symmetry between right and left sides. This can affect any part of human body or whole^[1]. On facial skeleton there is slight difference between right and left side which taken into account as normal and usually it remains unnoticed but deviation from marked proportionality is of great concern esthetically, anatomically and physiologically^1,[2]. Studied data shows minor asymmetries with right hemiface wider than the left side with concomitant chin deviated towards left¹. On face, mandibular asymmetries are most commonly noticed.Their causes are divided into four major groups as:developmental,traumatic,pathological and functional.Descriptive data by Profit and Severt reported it as 5% in upper face,36% in middle third, and 74% in lower third. Their clinical evaluation included lower third of face, dental midline relationship between maxilla /mandible, gonial angle on both sides, mandibular lower border, gingival display both anterior/posterior, open bite, deep-bite, maximal inter-incisal opening and temporo-mandibular joint symptoms.

Cleft lip and palate clinically represents usually as mid face deficiency, posterior crossbite with class III molar relationship. Available evidance on mandibular

asymmetry related to cleft lip and palate  is conflicting^1,2,3,4. Morphological differences consistently coexists between unilateral and bilateral cleft lip and palate.In bilateral cleft lip and palate due to forward displacement of premaxilla there is no shortening  of total depth of upper jaw.This found in contrary positive in unilateral cleft lip and palate.However both due to marked backward displacement of maxilla shows maxillary retrusion.The total length of face is increased in bilateral cleft lip and palate with more posterior growth rotation.In unilateral cleft lip and palate the total length of face is decreased in bilateral cleft lip and palate there is more retroclination of palate with retrusion of mandible^2,3,4,5,6. It is also being studied that condylar height found to be significant(p<.05) in this group which later discussed to be as significant factor related to symmetrical posterior vertical height of mandible.Further there is more posterior displacement of both zygoma and orbit in this group.Because of increased retrusion of mandible the skeletal profile of bilateral cleft lip and palate is not flattened as compared to unilateral cleft lip palate^2,4,5. This is also associated with increase protrusion of premaxilla.Upper incisors are more retroclined in bilateral cleft lip and palate as compared to unilateral cleft lip and palate^2,3. Overall in unilateral cleft lip and palate asymmetry found to be more on cleft side then non cleft side .

According to data from the international perinatal database of typical oral clefts the total prevalence of cleft lip with or without cleft palate is 9.92 per10, 000 live births. This includes cleft lip3.28 per 10,000.Cleft lip and palate was 6.64 per 10,000.76.8% were isolated,15.9% had systemic malformations and7.3% associated with syndromes⁴.Cleft lip and palate not associated with any syndromic conditions are caused by an intrinsic combination of both genetic and environmental factors.This termed to be multifactorial.The developing embryo inherits genes that increases the risk of cleft lip and/or palate; such genes upon exposure to certain environmental factors later develops into cleft⁵.

In Pakistan prevalence of cleft lip and palate is1per 523births  among which cleft lip is42%, cleft palate 24 %,and combine cleft lip and palate is 34%.Boys predominates with cleft lip and combine lip/palate while girls predominates with cleft palate only⁶.

Overall reported cases in our tertiary care hospital includes patients from different regions of country with varying severity of cleft types both syndromic and nonsyndromic.Due to growth variability among both unilateral and bilateral cleft lip and Palate group there suggestive to be a consistant chance of developing facial asymmetry. Facial asymmetry is majorly evident in lower face skeleton where mandibular anatomy, morphology and position in three dimensional planes plays crucial part which later significantly affects treatment planning, decision and outcome including orthognathic surgical procedures. Therefore, our primary aim is to compare mandibular vertical asymmetry between cleft lip palate and normal subjects.

OBJECTIVE

To determine the mean difference in mandibular vertical asymmetry between cleft lip/palate and normal class I occlusion subjects in Pakistani population.

METHODOLOGY

Pretreatment panoramic radiograph of subjects were taken attending department of orthodontics at Dr.Ishratul-Ebad Khan Institute Of Oral Health Sciences[Dow University Of Health Sciences],a major tertiary care centre in Karachi-Pakistan.The sample for this study consisted of total 64 subjects divided into two groups:Group1consisted of 32 subjects with repaired cleft lip and palate[16complete unilateral and 16complete bilateral cleft lip and palate].Group 2 consisted of 32 subjects with normal facial morphology and class I occlusion(both males and females).Informed consent was taken from all patients.

Inclusion criteria for cleft lip and palate group was non-syndromic subjects of either gender presenting with repaired unilateral or bilateral cleft lip and palate. Exclusion criteria included Cleft lip and palate subjects with systemic diseases,incomplete repaired palate,open fistulas,developmental or acquired craniofacial muscular deformities,autoimmune conditions,syndromes,endocrine abnormalities,neurological problems,any previous history of orthodontic treatment and signs and symptoms of temporomandibular disorders.

Inclusion criteria for normal class I subjects included skeletal base class I[ ANB=2°±2°,SNA=82°±2°,SNB=78°± 2°], permanent dentition  with Angle’s molar classification I belonging to either gender.

Exclusion criteria included significant medical history, history of trauma or any previous treatment and surgery, impacted, missing teeth,periodontally involved teeth,subdivision molar classification,skeletal base II and III with molar class I.

Mandibular Asymmetry Index[condylar,ramal, condylar plus ramal] and gonial angle measurements were calculated on dental panoramic radiograph as depicted by Habets, et al(figure 1)^7,8,9. Condyle and ramus of both right and left side was drawn on cephalometric lead acetate sheets from OPG Land marks were identified as Aline connecting the most lateral point of condylar image01and ascending ramus marked as image 02.The

Figure 1:Showing Land marks identified as A line connecting the most lateral point of Condylar image 01 and Ascending Ramus image as 02.The distance between both indicated as Ramal height[RH]. Ramal tangent from the superior most point of the Condyle image.A perpendicular line drawn from tangent above called B line.Condylar height[CH]measured from B line on A line to the 01 point.Gonial angle[GA] measured as angle formed by lines tangent to the posterior ramus and inferior border of mandible.

distance between both was indicated as ramal height[RH].Ramal tangent was taken from the superior most point of the condyle image.A perpendicular line was drawn from tangent above called B line.Condylar height[CH] was measured as from B line on Aline to the 01 point.Gonial angle[GA] was measured as angle formed by lines tangent to the posterior ramus and inferior border of mandible.Asymmetry indices value calculated above 3% were indicated to be as true mandibular posterior vertical asymmetry.Asymmetry indexes were calculated by following formula as:

For Condylar Asymmetry Index:
[(CHRIGHT-CHLEFT)/CHRIGHT + CHLEFT)] X100

For Ramal Asymmetry Index:
[(RHRIGHT-RHLEFT)/RHRIGHT+RHLEFT)]X100

For Condylar+Ramal Asymmetry Index:
[(CO+RH) RIGHT-(CO+RH)LEFT)/(CO+RH)LEFT)
/(CO+RH)LEFT)/(CO+RH)LEFT)/(CO+RH)RIGHT+
(CO+RH)LEFT)]X100.

For reliability of assessment sufficient care was taken with head positioning to avoid any vertical and angular measurement errors.This signify the limit of head positioning in all direction altogether with occlusal plane not tilted more than 10 degree.

STATISTICAL METHODS

Statistical analysis was done by using SPSS for windows version 16. ANOVA and Mann -Whitney U test was used to calculate intergroup comparison. Paired t-test was used to calculate differences between cleft to non cleft side in unilateral cleft lip palate group. All Statistical analysis were calculated at 95% confidence level with significant P-value<.001.

RESULTS

Calculated descriptive statistical measurements between normal occlusion and cleft lip and palate group shows significant differences (P<.001) in both condylar and ramal asymmetry index.But over all intergroup comparison for condylar plus ramal height asymmetry Index did not show any significant result (P>.05). While amongst the unilateral cleft lip and palate this shows both ramal height and gonial angle at cleft side to be statistically significant (P<.001). A statistical measurement shows no significant difference for gender. Results are also shown in tables I, II, and III respectively below:

DISCUSSION

Symmetry in mandibular morphology is key to aesthetics which compounds to the whole face both

aesthetically and developmentally.Asymmetrically developed mandible significantly affects the facial lower third along with the developing nasomaxillary complex.Varying degree of asymmetry was found in cleft lip and palate as described by Laspos, et al,Smahel and Mullerova, et al.^10,11,12. Overall world wide data collected on mandibular asymmetry in unilateral and bilateral cleft lip and palate is limited.Mandibular asymmetry in unilateral and bilateral cleft lip palate is inconsistent with cleft type. Developmental growth studies shows variation in relation to the craniofacial development due to differences in patterning,timing and variability factors.Genetical influences encompasses the overall period and hence rendering them unpredictable.This outcome describes physiologically enveloped adaptive mechanism.

Mandible makes up the 95% of lower facial skeleton.Lower face is also under developmental influences of cranial base and temporal fossa.Ishiguro, et al and Athanasiou, et al.,described in their study about increase in mandibular breadth.This in turn found to have direct association with mandibular fossa  making them both directly proportion to each other^13,14. Also Bjork, Skieller, et al.,comprehensively discussed about developmental effects related to the cranial base displacement, glenoid fossa remodeling, and condylarramal relocation with growth¹⁵. Melnick et al.,

studied the difference between cleft and non cleft in longitudinal growth study and found no more than 0.5mm of difference.¹??Our study shows over all mean difference in condylar height plus ramal height index not more than 3%.

Both unilateral and bilateral cleft lip and palate have also suggested possible association with number of syndromes like for example: Goldenhar, mandibulofacial dysostosis syndrome, craniosynostosis syndromes; which readily affects both cranial base and temporal region anatomy thus significantly affecting the symmetry of lower face skeleton.On contrary this is not consistent with those who do not have an association with syndromes.In individuals either with unilateral and bilateral left lip palate having no syndromic association there may found to have an associated lower face skeleton asymmetry but not to the statistically marked significant level.Our study also did not find any significant(P<.05) mandibular asymmetry as overall condylar plus ramal asymmetry index between normal and non syndromic cleft lip and palate group shows insignificant results(P>.05). Asymmetry Indices value should be above 3% to be indicated as true mandibular posterior vertical asymmetry⁸.

It has been described variably that mandibular growth is majorly influenced by pterygoid muscle trauma due to surgery, inhibited maxillary growth and affected functional changes.But this had later found to be unaffected. Omar et al., in his 204 patient sample comparative study including complete unilateral cleft lip palate, complete unilateral lip and alveolus, and isolated palate reported no statistically significant changes related to mandibular growth².

Dental panoramic radiograph has remained the method to evaluate mandibular asymmetry as explained by Habets et al,Miller and Smidt,kurt, et al and Uysal, et al.In descriptive study of unilateral cleft lip palate by kyrkanides and Richter et al.,the degree of antigonial notching is significant factor found in mandibular and lower facial asymmetry(P<0.001)^7,8,17. Jena et al; explained in their unilateral cleft lip and palate subjects that with less severe maxillary sagittal hypoplasia there found to have a statistically significant differences in both ramal and condylar plus ramal height.Further gonial angle was also significantly increased.In our study dental panoramic radiograph was taken with caution without altering head position and occlusal plane^{17,18,19,20,21,22,23}. It shows that both the ramal height and gonial angle found to be increased with cleft side among unilateral cleft lip and palate group.This is indicative of glenoid fossa remodeling,condylar-ramal relocation and growth.Comparative study between cleft and noncleft side among unilateral cleft lip and palate by Horswell,Levant.,also concluded statistical significant difference( P<.05) in gonial angle.It was found to be increased on cleft side which later explained to be a compensatory mechanism of mandible on cleft side^24,25. Our findings found to be inconsistent to this. Laspos,et al.,described in their study on PA Cephalometry that asymmetry of ramal height in unilateral cleft lip and palate was significant¹⁰. Their mean differences shows no significance (p>.05) between the non cleft and cleft group. Our study also shows similar findings in this regard.

Altogether both cranial base and temporal region anomalies may involved in lower face skeleton asymmetry. Descriptive study by kurt et al.,found only difference in condylar height which was statistically significant (P<.05) in bilateral cleft lip palate group¹⁷.This might suggestive of variation in one unit condylar -ramal relocation along with the developmental distinct pattern of both bone and muscles in bilateral cleft lip and palate group.

CONCLUSION

Mandible in both non syndromic unilateral and bilateral cleft lip/palate subjects was found to be similar to normal subjects. However there was a difference in condyle, ramus and gonial angle between cleft and noncleft side in unilateral cleft lip and palate subjects.

REFERENCES

1. Severt TR, and Proffit WR.The prevalence of facial asymmetry in the dentofacial deformities population at the university of North Carolina.Int J Adult Orthodon Orthognath Surg.1997;12:171-176.
2. Omar, et al.Mandibular Morphology and Spatial Position In Patients With Clefts:Intrinsic or Iatrogenic.Cleft Palate Craniofac J.1992;29:369-375. 3.
3. Krogman, et al.Sex differences in craniofacial growth from one month to ten years in cleft lip and palate.Cleft Palate J.1982;19:62-71.
4. IPDTOC Working Group.Prevalence at birth of cleft lip with or without cleft palate:data from the international perinatal database of typical oral clefts (IPDTOC).Cleft Palate Craniofac J.2011;48:66-81.
5. Lindral AC, et al.Genetic factors and orofacial clefting.SeminOrthod.2008;14:103-114.
6. Elahi MM, et al.Epidemiology of cleft lip and cleft palate in Pakistan.Plast Reconstr Surg.2004;113:15481555.
7. Habets, et al.The orthopantomogram, an aid in diagnosis of temporomandibular joint problems.I.The factor of vertical magnification.Journal of Oral Rehabilitation.1987;14:475-480.15:465-471.
8. Sezgin, et al.Mandibular asymmetry in differentmalocclusion patterns.Angle Orthodont. 2007;77:803807.
9. Laspos, et al.Mandibular asymmetry in noncleft andunilateral cleft lip and palate individuals.The Cleft Palate Craniofac J.1997;34:410-416.
10. Smahel Z, and Brejcha M.Differences in craniofacialmorphology between complete and incomplete unilateral cleft lip and palate in adults.The Cleft Palate Journal.1983; 20:113-127.
11. Smahel Z, and Mullerova Z.Craniofacial morphologyin unilateral cleft lip and palate prior to palatoplasty.Cleft Palate J.1986; 23:225-232.
12. Athanasiou, et al.Frontal cephalometric evaluationof transverse dentofacial morphology and growth of children with isolated cleft palate.J Craniomaxillo fac Surg.1991;19:249-253.
13. Ishiguro, et al.A longitudinal study of morphologicalcraniofacial patterns via PA xray head films in cleft patients from birth to six years of age.The Cleft Palate Journal.1976;13:104-126.
14. Bjork A, and Skieller.Normal and abnormal growthof mandible. a synthesis of longitudinal cephalometric implant study over a period of 25years. Eur J Orthod.1983;5:1-46.
15. Melnick, et al.A cephalometric study of mandibularasymmetry in a longitudinally followed sample of growing children.Am J Orthod DentoFac Orthop.1992; 101:355-366.
16. Kurt G, et al.Mandibular asymmetry in cleft lip and palate patients.Eur J Orthod.2010;32:19-23.
17. Kambylafkas, et al.Validity of panoramic radiographsfor measuring mandibular asymmetry.Angle Orthodont. 2006; 76:388-393.
18. Kurt G , et al.Mandibular asymmetry in class II subdivision malocclusion.Angle Orthodont .2008;78:3237.
19. Kyrkanides S,and Richter L.Mandibular asymmetry and antegonial notching in individuals with unilateral cleft lip and palate .The Cleft Palate Craniofac J.2002;39:30- 35.
20. Kirkanidis, et al.Skeletal asymmetries of the
21. nasomaxillary complex in non cleft and post surgical unilateral cleft lip and palate.Cleft Palate Craniofac J.1995;32:428-433.
22. Kirkanidis, et al.Asymmetries of upper lip and nose in non cleft and post surgical unilateral cleft lip and palate individuals.Cleft Palate Craniofac J.1996;33:306311.
23. Jena, et al.Effects of sagittal maxillary growth hypoplasia severity on mandibular asymmetry in unilateral cleft lip and palate subjects.Angle Orthodont. 2011;81:872-876.
24. Horswell B, and levant B.Craniofacial growth in unilateral cleft lip and palate: skeletal growth from eight to eighteen years. Cleft Palate J.1988; 25:114-121. 25. Shetye PR , and Evans CA.Midfacial morphology in adult unoperated complete unilateral cleft lip and palate patients .Angle Orthodont. 2006;76:810 816.

1. Post Graduate Fellowship Residency completed. Orthodontics Department. Dr.Ishratul-Ebad Khan Institute Of Oral Health Sciences-Dow University Of Health Sciences. Karachi-Pakistan.
2. Head of Department and Associate Professor. Orthodontics Department. Dr.Ishratul-Ebad Khan Institute Of Oral Health Sciences-Dow University Of health Sciences. Karachi-Pakistan.
3. Associate Professor. Orthodontics Department. Dr.Ishrat-ul-Ebad Khan Institute Of Oral Health Sciences-Dow University Of health Sciences. Karachi-Pakistan.
   Corresponding author: “Aiyesha Wahaj ” < draiyeshawahaj@yahoo.com >

Samira Adnan¹                                BDS

Farhan Raza Khan²                      BDS, MSc, MCPS, FCPS

INTRODUCTION

Avulsion is defined as the complete displacement of tooth out of its socket. This kind of dental trauma causes the periodontal ligaments to be severed with or without fracture of the alveolus. (1, 2) Reported incidence of dental avulsion is 1-11% of all dental injuries to the permanent dentition, with the maxillary central incisor being the most frequently involved tooth. The age group of 7-10 years appears to be most affected. (3) Detrimental consequences for the avulsed tooth as a result of this type of trauma include a severed vascular and nerve supply, resulting in the death of the pulp, especially in a tooth with a closed apex. More importantly, the complete separation of the tooth from the socket results in tearing of the periodontal ligaments, leaving viable periodontal ligament cells on the root surface.(4) The periodontal ligament cells, a quarter of which comprise of PDL fibroblasts, are responsible for the re-attachment of the avulsed tooth once it is re-implanted into its socket. Although immediate re-implantation is the treatment of choice as it results in PDL healing in up to 85% of mature teeth (5), it is not always possible to immediately re-implant the avulsed tooth. Therefore, an intermediate storage or transport media is essential for storage of such a tooth until the time it is re-implanted. The prognosis for success of reimplantation attachment is greatly dependent on the extra-oral dry time to which the avulsed tooth is exposed and placement in a storage medium that is capable of maintaining the viability of the PDL cells.(6) Numerous properties of the ideal storage media have been described in literature(7) (Table 1), and the pursuit for the best

Table 1. Properties of the ideal storage media storage medium has yielded a vast array ofresearch on various

substances. This article attempts to provide an overview of the multiple types of storage media reported in literature with a brief account of their suitability as storage or transport medium. The storage media are listed according to their origin and composition.

Tap water:

Use of tap water to store avulsed teeth is not recommended as it is not compatible with PDL cells because of its hypotonic osmolarity which causes cell lysis, and is reported to causes replacement resorption in avulsed teeth when they are place in it. (8) It is considered the least desirable storage medium.

Saliva:

Saliva is a readily available, natural storage medium. Despite this fact, due to the presence of substances like enzymes and bacteria and its non-physiologic osmolarity, which can exert harmful effects on the PDL cells, this can at best be used as an interim storage medium (no longer than 30 minutes).(8, 9) If stored for more than 60 minutes, a significant decrease in functional capacity of PDL cells occurs.(10)

Milk and Variants:

The use of milk as storage media has gained much popularity due to its ease of availability, cost effectiveness and physiologic osmolarity. Studies in literature have investigated various forms of milk including whole milk, skimmed milk, low fat content milk, baby formula and long shelf life milk. With a pH of 6.5-7.2 and the presence of essential nutrients important for maintaining the viability of PDL cells, milk can be considered as acceptable storage medium in most situations(11), increasing the life of the PDL cells on the root surface.(12) Milk can maintain the viability of PDL cells from 2hrs(13) to 6 hours.(8, 10, 14-17) Milk that has been refrigerated or which has a lower fat content demonstrated better results as storage media in various studies.(10, 15-18) Its clonogenic and mitogenic capacity for PDL cells is considered equivalent to Hank’s Balanced Salt Solution [HBSS].(19, 20) Some of the drawbacks of using milk as storage medium are the presence of antigens that could interfere with the process of reattachment of PDL cells when the tooth is re-implanted.(7) Also the milk needs to be fresh and refrigerated.(21) Sour milk should not be used as it is considered to be harmful.(10)  Milk also has no proven role in assisting cell mitosis in PDL cells.(8, 14)

Soy Milk:

Soy milk, the water extract of soybean, contains no cholesterol or lactose and very small amounts of saturated fatty acid .(22) It is considered an excellent culture medium forcell growth and biochemical activities. Recent studies have shown that soy milk in contact with periodontal ligament cells promoted good cell viability, comparable to HBSS and milk and hence is recommended as a storage medium.(22, 23)

Saline:

Although isotonic saline has been used in various studies for its effect as a storage medium, it is unable to maintain the metabolism of PDL cells. It has comparable osmolality to the PDL cells, but lacks nutrients. It is considered acceptable to place an avulsed tooth in isotonic saline rather than storing it dry(24)although in another study, no significant difference was found in the development of ankylosis between teeth kept dry or placed in normal saline.(25) It is acceptable to place avulsed teeth in this storage medium for not more than 10 minutes.(26)

Oral Rehydration Solutions:

Ricetral is a commercially available oral rehydration formulation, consisting of essential nutrients like glucose and vital salts which help in maintaining cell metabolism. They are marketed in sealed sterile pouches and easily available over the counter in addition to being economical. It does not promote cell mitosis and regenerative capacity of the PDL fibroblasts. Its ability to maintain PDL cell viability was demonstrated to be equal to HBSS in a study, both retaining PDL vitality better than milk.(27)

Hank’s Balanced Salt Solution:

HBSS is a salt-solution which is pH balanced and contains essential metabolites needed for viability of PDL cells. It is considered a gold standard for storage media used in transport of avulsed teeth and it is used to compare the efficacy of other storage media. Its ability to maintain the vitality, clonogenic and mitogenic capacity

of PDL cells for up to 48 hours has been proven.(8) It is considered superior to many other media in this regard (11, 20, 28) and can be used to store the avulsed tooth for at least 24 hours. It has also shown to replenish metabolites which have been depleted from PDL cells.(13) Hence, it has been recommended to place avulsed teeth in HBSS for 30 minutes before reimplantation in order to replenish the PDL cells, even if the avulsed teeth have been stored in an appropriate storage medium. HBSS is not readily available to public as its use is more in research laboratories, although in some countries it is available in emergency kits [SaveA-Tooth, PA, USA]. This kit comprises a small basket to hold the avulsed tooth while it is submerged in HBSS, until the tooth is re-implanted. Lack of availability and cost are considered the major draw-backs for this storage medium.

Propolis:

Propolis is a resin obtained from conifer trees. This sticky material is used by bees for constructing and maintaining their hives. It is a non-toxic biological material with anti-inflammatory, anti-bacterial, antioxidant, anti-fungal and tissue regenerative properties.(29) The main ingredient of propolis are flavanoids.(30) Its chemical composition can be highly variable due to the different variety of plants the honey bees can visit while collecting this material. Recent studies have shown 10% propolis to be an effective storage media when compared with milk, HBSS, tap water and DMEM.(31)  Due to the fact that propolis is not readily available, its utility as a storage media is diminished when compared to other ready available materials.

Coconut Water:

Coconut water is a biological liquid which is pure, sterile and rich in nutrients like amino acids, proteins, vitamins and minerals. Readily available in tropical countries, it is an isotonic solution which can be obtained fresh directly from coconuts or commercially in packages and bottles. When compared with other media like HBSS, propolis and milk, it was found that coconut water was the most effective in maintaining viability of PDL cells.(32) The same study found the combination of coconut water with sodium bicarbonate to be more effective but some studies have also demonstrating contradicting results.(33) Since the pH of coconut water is 4.1, it has harmful effects on cell metabolism until sufficiently neutralized.(33) Further research in this regards needs to be undertaken before coconut water can be used effectively as a storage medium.

Egg white:

This medium has not been found to be significantly different than HBSS in some studies in terms of cell viability and demonstrates greater PDL healing when compared with milk.(34) It can be used to store avulsed teeth for up to 10 hours.(35) One study found no difference between milk, egg white and artificial saliva.(36) Although it is easily available, its major setback is impracticality of use.

Salvia officinalis:

Salvia officinalis is a perennial, evergreen shrub with blue to purplish flowers. It is a member of the family Lamiaceae and is native to the Mediterranean region; it has a long history of medicinal and culinary use. The extract from this plant has been used as spasmolytic, antiseptic and astringent.(37) This extract has been proposed as a storage medium for avulsed teeth because of the anti-oxidants effects caused due to the presence of its phenolic components like rosmarinic acid, camosic acid, salvianolic acid and derivatives.(21) These antioxidants help to prevent root resorption by inhibiting the effect of osteoclastic cells. Studies have shown that Salvia extract at 2.5% helps maintain PDL cells viability over longer periods of time (3, 6, 12 or 24 hours) when compared with HBSS, phosphate buffered saline and tap water.(21) It has also demonstrated anti-microbial and anti-inflammatory properties. Thus salviaofficinalis can be recommended as suitable storage media for avulsed teeth.

Morus rubra:

Morusrubra [red mulberry] belongs to the Moraceae family and active components include flavonoids, alkaloids an polysaccharides.(38) In a study(39), Morusrubra juice at 4% concentration was found to be superior to HBSS for maintaining PDL cell viability for upto 12 hours.

Epigallocatechin-3-gallate [EGCG]:

Epigallocatechin-3-gallate [EGCG] is a major polyphenol of green tea, is known to have various biological effects such as anti-oxidative, anti-carcinogenic, anti-mutagenic, anti-inflammatory, anti-microbial, and anti-viral activities.(40) Recently, research has been conducted to determine its role as an adequate storage medium. Greater viability of PDL cells of guinea pig and Beagle dog has been maintained when placed in EGCG. (41, 42) A study on extracted human teeth showed that EGCG can be used adequately as a storage medium, with a higher potential than HBSS and milk to promote favorable reimplantation, with less risk of root resorption and ankylosis. (43)

CaesinPhosphopeptide:

Casein phosphopeptides [CPP] are derived from casein, which account for 80% of the total protein in bovine milk.(44) They can form soluble organophosphate salts and may function as carriers for different minerals, especially calcium. Their role in preventing demineralization and aiding in remineralization has been demonstrated.(45) In a study which investigated the use of different concentration of commercial CPP-amorphous calcium phosphate as storage media by observing morphological changes in fibroblast cells, it was found that cell apoptosis did not occur when very dilute concentrations of CPP-ACP [10-12] was used as storage medium(46) and further research was deemed necessary to determine the ideal concentration for preserving PDL cell viability.

Conditioned medium:

This medium is derived from supernatant of human gingival fibroblasts grown in culture. Since this medium contains stimulatory growth factors produced by the gingival fibroblasts, it is believed to have a beneficial effect on the PDL cells and their proliferation.(21) This medium is also not readily available for general use, which limits its practicality.

Culture media:

Culture media can include Eagle’s medium, alphaMinimum Essential Media and alpha-MEM-S( with addition of foetal calf serum and antibiotics).(7) Eagle’s medium contains many nutrients like amino acids, vitamins and bicarbonates considered essential for maintaining the viability and proliferative capacity of PDL cells for longer time periods when compared with other storage media (48-53 hrs).(47) The addition of growth factors like platelet derived growth factor, insulinlike growth factor, epidermal growth factor and many others, helps to enhance the clonogenic and mitogenic capacity of PDL cells for longer periods of time.(48) Recently, research has demonstrated that the use of special cell culture medium (SCCM), which has been formulated especially to be used as a storage medium for avulsed teeth, is better at maintaining PDL cell viability than HBSS for time period of longer than 24 hours.(49) Another variation of Eagle’s Modified Essential Medium (EMEM) is Dubelco’s modified Eagle’s Medium (DMEM) which contains a greater concentration of vitamins and amino acids as well as glucose compared to the EMEM.(21) Despite their excellent properties, due to their need for refrigeration and lack of availability, all these culture media are not considered as practical for use as storage medium for avulsed teeth.

Custodial:

This medium is the registered trademark of Dr. Franz. It contains a histidine-tryptophan ketoglutarate solution containing high flow properties and low potassium content.(21) It is basically an organ transport medium, also used for perfusing and flushing donar organs prior to their removal. In a study it was reported that custodial was comparable to HBSS in terms of cell viability.(50) Similar to other organ storage medium, it is not available to public which limits its practicality as a storage medium for avulsed teeth.

Via Span:

This is a medium which was formulated for use in transplant procedures. It is used for cold storage of organs when they are removed from a donor. It is clear to light yellow in color, sterile and non-pyrogenic, with a pH of 7.4.(21) It has shown to maintain the viability of PDL cells effectively(11), while maintaining cell morphology.(51) Drawbacks of using Via Span include the need for refrigeration, high cost and inaccessibility.(51)

Dentosafe:

Dentosafe (Miradent, Germany) is the commercial name of a tooth rescue box containing Special Cell Culture Medium (SCCM) which is a combination of amino acids, vitamins and glucose.(21) In the USA it is marketed as EMT Tooth Saver (Phoenix, USA). It has demonstrated the maintenance of vitality of PDL cells for 48 hours at room temperature.(47) If unopened, this medium has a shelf-life of 3 years.  A study by Pohl et al (52)showed that avulsed teeth placed in Dentosafe solution showed functional healing and recommended that Dentosafe should be included in all first aid kits, especially in locations prone to tooth avulsion injuries like schools, sports ground and facilities, public pools as well as emergency units like hospitals and ambulances. The use of this system is self-explanatory and simple to understand for lay persons. Although effective, this medium is still not easily available in many countries.

Contact lens solutions:

Since contact lens solutions are basically saline solutions, there use as storage media has been researched in some studies.(28, 53) However when compared with other storage media, they were deemed to be harmful and thus are not recommended for storing and transporting avulsed teeth.(14)

Gatorade:

This drink was originally developed for athletes in order to replenish electrolytes lost during exercise and physical activity. Compared to tap water, the use of Gatorade as storage medium yielded better results for PDL cells survival.(16) Although it is relatively easily available at sporting events, where avulsion injuries to teeth tend to occur, its osmolarity causes cells destruction and hence it is not recommended for long term storage of avulsed teeth.(21)

CONCLUSION

Although research has been undertaken on a wide variety of materials to be used as storage media for the transport of avulsed teeth, lack of availability and high cost limit the use of majority of these media. Therefore, because of its acceptable performance, ease of availability and lesser cost, milk remains the storage medium of choice in cases where avulsed teeth cannot be immediately reimplanted. This knowledge should be made part of a public awareness program, in order to properly handle and save a large number of avulsed teeth, the prognosis of which can greatly improve if placed in an appropriate storage medium.

REFERENCES

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1. Resident Operative Dentistry Program Dental Section, The Aga Khan University Hospital, P.O.Box 3500, Stadium Road Karachi, 74800 Pakistan.
2. Assistant Professor, Operative Dentistry Dental Section, The Aga Khan University, P.O.Box 3500, Stadium Road Karachi, 74800 Pakistan.

Corresponding author: “Dr. Samira Adnan” < samira.adnan@aku.edu >

Kiran Rehman¹                                BDS, FCPS

Robia Ghafoor²                               BDS, FCPS

Farhan Raza Khan³                      BDS, MSc, MCPS, FCPS

INTRODUCTION

The dental pulp is a neurovascular tissue that resides in a rigid chamber comprising of tooth enamel dentine and cementum. This rigid encasing not only provides mechanical support but also protects the pulp from biochemical insults and against microbial invasion as well. Therefore, it is important that the tooth structure maintains its structural integrity so that the dental pulp remains protected. Caries, cracks, fractures and open restoration margins provide pathways for microorganisms and their toxins to enter into the  pulp.(1)

Toothache is one of the most common complaints seen in the dental clinics, and yet diagnosis of pulp disease is often difficult due to the seemingly unclear symptoms(1) Even in the presence of a detailed history and a thorough clinical examination, its  diagnosis can be difficult, and can be further complicated by presence of pathology in surrounding anatomical structures and referred pain. A delay in diagnosis can lead to a delay in treatment therefore causing undue distress to the patient.

Non odontogenic pathology is sometimes difficult to diagnose and appears clinically as odoontogenic toothache, andvice versa. Especially if present in the vicinity of roots of teeth, and therefore the clinician may decide to defer treatment until localization of symptoms to be sure of odontogenic versus non odontogenic pain. Cracked teeth most often present with vague symptoms of pain and tenderness, reversible or irreversible pulpitic symptoms and are one of the more diffucltut entities to diagnose especially if present as partial or hidden cracks.

CASE REPORT

A 49 year old female presented to the Dental Clinics at Aga Khan University Hospital Karachi on August 15^th 2011 with complaint of mild tenderness in the upper right premolar area on chewing since two weeks. There was no history of spontaneous pain or sensitivity to hot, cold or sweets. She also complained of heaviness in the right maxillary sinus area.

Her past medical history revealed that she had a history of chronic sinusitis, and took anti-allergic medication for allergies. She had a history of acute bronchitis in 2003, and was hospitalized in 2010 for dengue fever.

On her extra-oral examination, there were no facial asymmetries; mild tenderness on palpation in the right maxillary sinus area was noted. No palpable lymph nodes and no tenderness over the temporo-mandibular joint and muscle area were noted.

Intra oral examination revealed short clinical crowns, more square in shape. No carious lesion was seen. However all her third molars and left upper and both lower first molars were missing.  . The upper first molars # 26, 36 and 46 were extracted when she was 12 years old due to dental decay. She never got replacements done for these teeth. There was no tenderness on percussion in any of the teeth except  teeth # 14, 15 and 16. Periodontal probing with BPE revealed a score of 1 in all sextants. There was no mobility seen in any tooth. Vitality testing of teeth # 14,15 1nd 16 was carried out with endo ice, warm gutta percha sticks and electric pulp testing. All methods revealed vitality status within physiological limits.

Radiographic examination revealed no abnormal findings; the lamina dura of the teeth in question was intact with adequate crestal bone levels. However, some calculus spurs were identified.

Figure 1: Radigraph at the time of presentation

DIAGNOSIS

A definitive diagnosis could not be reached at that point in time, her occlusal contacts on teeth #13, 14 and 15 were adjusted, full mouth scaling and polishing was carried out and she was advised to come back for a follow up after 3 weeks time. She was also advised to consult with the ENT specialist for management of chronic sinusitis.

The patient consulted with ENT and was advised a Para-nasal Sinuses Radiograph (PNS view) and a cystic lesion in the right maxillary sinus area was identified. (See figure 2) She was prescribed antibiotics and nasal

Figure 2: PNS view advised by the ENT Specialist

decongestants and surgical drainage and resection if the symptoms did not subside.

The patient did not return for a follow-up until December 2011 with acute pain in her right maxillary first premolar area. She reported of worsening symptoms during the past four months with a decrease in her chewing efficiency on the affected side for which she was on multiple courses of antibiotics advised by her ENT and general physician. Periapical radiograph was repeated of tooth #13,14 and 15 and an apical radiolucency with tooth #14 was evident (Figure 3). The tooth was acutely tender to palpation, thermal testing

Figure 3: Peripaical radiograph 4 months after initial presentation

was repeated and the tooth was not responsive to any of the stimuli (cold, hot, EPT).

Mobility of this tooth was within normal limits (2mm) and periodontal probing depth was 3.5 mm.

The diagnosis made was Pulp necrosis secondary to crack tooth syndrome and the treatment plan advised was root canal treatment of tooth #14 followed by a crown, full mouth debridement and replacement of missing teeth by fixed or removable prostheses.

Root canal treatment was carried out and completed after which the symptoms resolved completely in 3 weeks’ time.

Figure 3: Immediate post operative image

3 months follow-up

DISCUSSION

Periapical lesions can be of endodontic or nonendodontic origin and may involve one or multiple adjacent teeth. Lesions of endodontic origin have been classified as periapical abscesses, granulomas, and cysts depending on their histological typing (2).  Periapical lesions of non-endodontic origin can develop around the teeth’s periapical region and if not diagnosed early and correctly can expand to large lesions. Reports about nonendodontic lesions mimicking apical periodontitis and their misdiagnosis can be found frequently in the literature. (2)

Diagnosis is confirmed with the help of a detailed history and examination, periapical radiographs and electric and thermal pulp testing. However, two dimensional periapical radiographs often present with limitations in assessment of three dimensional root structures.  Cone beam computed tomography is a better aid to reach a diagnosis in complicated cases and particularly could have added sufficient value in this case by disclosing any cracks or fractures and also in showing the link of the cystic lesion to the tooth. In this particular case, the first evaluation did not reveal any radiographic changes nor was the history or clinical examination guiding the clinicians toward a diagnosis In this particular case, the first evaluation did not reveal any radiographic changes nor was the history or clinical examination guiding the clinician towards a diagnosis. The patient had a history of chronic sinusitis, and therefore, was advised to seek an ENT evaluation as well. The patient did not return to clinic till 4 months after, when there was an abscess formed associated non vital # 14 which was extremely tender to palpation on presentation.

In this authors’ opinion, had the patient returned to clinic within 3-4 weeks of first evaluation, she would have been saved multiple visits to ENT, numerous courses of antibiotics and a visit to the emergency room as well. The question remains, whether it was lack of clinical judgment or lack of substantial evidence responsible for not formulating a definitive diagnosis earlier, or whether the endodontic pathology was below the diagnostic threshold on first presentation. In any case, the patient did not show up for 4 months after first examination till she got her upper premolar affected with abscess.

Infected root canal systems can lead to inflammation of periapical alveolar bone therefore leading to the development of apical periodontitis. When associated with maxillary canines and premolars, apical periodontitis can trigger inflammation of the mucosal lining of the maxillary sinus and lead to development of maxillary mucositis, periositits as well as sinusitis. Similarly, maxillary sinusitis can also present with symptoms of toothache and tenderness in the area of maxillary canines and premolars. This patient visited her

ENT frequently due to chronic sinusitis and kept getting treatment for such.

Other differentials that were suggestive of her symptoms were occlusal trauma leading to apical periodontitis, cracked tooth syndrome, and endoantral syndrome.

At her first visit, occlusal trauma was the most likely suspect due to multiple missing molars, mild pain only on chewing which lead to the dentist adjusted her heavy occlusal contacts on the affected side by means of superficial enameloplasty.

The normal mucosal lining within the maxillary sinus is <1 mm in thickness and not discernible radiographically. Sinus mucositis may develop in response to infectious or allergic stimuli producing a band of noncorticated mucosal tissue paralleling the bony wall of the sinus.(3-5) In some instances new bone formation is also seen when the noxious stimuli are odontogenic and travel through cancellous bone towards the corticated floor of the sinus.(3)

A diagnosis of endoantral syndrome could not be made at the second visit due to lack of communication and presence of corticated bone between the tooth and the floor of the right maxillary sinus.

The most likely diagnosis reached in the present case report was that of cracked tooth syndrome. Guersten et al. stated that ‘excessive forces applied to a healthy tooth or physiologic forces applied to a weakened tooth can cause an incomplete fracture of enamel or dentine. (6) Cracks are not discernible on radiographs, even on multiple angulations of exposure. Cone beam computed tomography, and infrared thermography (7) are currently better modality to diagnose thin hairline fractures in teeth. Due to lack of resources and the case being reported from a developing country, we did not have access to such advances in our practice.

In a recently published review Banerji has stated the commonly presenting signs and symproms of cracked tooth syndrome which include: (8)

• Sudden sharp pain on biting or releasing the bite
• Sensitivity to thermal stimulus
• Presence of symptoms from weeks to months
• Inability to localize the affected tooth or teeth
• Pain may be initiated by cuspal deflection or pressure, and/or tooth grinding
• Fracture lines may or may not be clinically discernible even with magnification, dyes and transillumination
• Vitality tests show a positive response at initial presentation
• Radiographs are usually inconclusive

Keeping all of the above signs and symptoms in mind, the most likely diagnosis reached for our patient was that of cracked tooth syndrome.

The patient was managed successfully with root canal followed by crown. The retention cyst seemed to resolve with time and intervention was not required for its management. In the authors’ opinion it was an endodontic abcess that appeared as a cystic lesion in the right maxillary antrum and was misdiagnosed as a retention cyst due to limitations of conventional 2dimensinal radiographic imaging techniques. The learning outcome of this case is that in a clinical situation where a clinician fails to reach a definitive diagnosis, the differential diagnoses should be discussed with the patients and assistance of other healthcare providers should be sought to offer a holistic care in multidisciplinary fashion. Lastly, the patient should be emphasized about meeting their scheduled appointments to avoid untoward outcomes.

RECOMMENDATION

In light of the case report it is advised to always keep the masked symptoms of cracked teeth in mind when diagnosis becomes a challenge. Also patients should be educated in detail about their symptoms and the pros and cons of keeping regular followups as advised by the clinicians.

CONCLUSION

The sooner the cracks are detected and treated the better is their long term prognosis. Cracked tooth syndomre needs more diligent observation and advanced diagnositic and treatment modalities to prevent propagation of cracks where teeth may be non salvageable.

REFERENCES

1. Yu C, Abbott PV. An overview of the dental pulp: its functions and responses to injury. Aust Dent J. 2007;52(1 Suppl):S4-16.
2. Suter VG, Buttner M, Altermatt HJ, Reichart Bornstein MM. Expansive nasopalatine duct cysts with nasal involvement mimicking apical lesions of endodontic origin: a report of two cases. J Endod. 2011;37 :1320-1326.
3. Nurbakhsh B, Friedman S, Kulkarni GV, Basrani B, Lam E. Resolution of maxillary sinus mucositis after endodontic treatment of maxillary teeth with apical periodontitis: a cone-beam computed tomography pilot study. J Endod. 2011;37:1504-1511.
4. Hauman CH, Chandler NP, Tong DC. Endodontic implications of the maxillary sinus: a review. Int Endod J. 2002;35:127-141.
5. Nimigean VR, Nimigean V, Maru N, Andressakis D, Balatsouras DG, Danielidis V. The maxillary sinus and its endodontic implications: clinical study and review. B-Ent. 2006;2:167-175.
6. Geurtsen W. The cracked-tooth syndrome: clinical features and case reports. Int J Periodontics Restorative Dent. 1992;12:395-405.
7. Matsushita-Tokugawa M, Miura J, Iwami Y, Sakagami T, Izumi Y, Mori N, et al. Detection of Dentinal Microcracks Using Infrared Thermography. J Endod. 2013;39:88-91.
8. Banerji S, Mehta S, Millar B. Cracked tooth syndrome. Part 1: aetiology and diagnosis. Br Dent J. 2010;208:459-463.

1. Assistant professor & Consultant, Operative Dentistry Liaquat National Hospital, Karachi
2. Senior Lecturer, Operative Dentistry Aga Khan University Hospital Karachi Karachi, PAKISTAN.
3. Assistant Professor, Operative Dentistry Aga Khan University, Karachi, PAKISTAN

Corresponding author: “Dr.Kiran Rehman ” < krehman83@yahoo.com >

Shahid Ali Mirani¹                                BDS, PhD

Feroze Ali Kalhoro²                            BDS, FCPS

Naresh Kumar³                                     BDS, PhD

Abdul Bari Memon⁴                            BDS, PhD

Faisal Bhangar⁵                                   BDS, FCPS

INTRODUCTION

Dental erosion (DE) is an acid-related loss of dental hard tissues that does not involve bacteria and is not induced with dental plaque. The etiological factors of dental erosion are divided into two groups, extrinsic and intrinsic factors . The dietary factors are the most common extrinsic erosive causes, which include acidic drinks, either pure fruit juices or carbonated soft drinks with added hydroxy organic and phosphoric acids. Time, type of drinks and its frequency of consumption have major influence in the development of an erosive tooth surface loss. In addition, bed time intake of fruit juices can cause severe tooth loss because of decreased salivary flow2, . The acids from dietary sources have distinct erosive potential. Citric acid has a significant destructive effect to teeth as it can chelate calcium in hydroxyapatite and form soluble citrates even after the pH rises. Some beers and herbal teas with low pH values have been reported to cause potential DE in vitro , . Frequent consumption of pickled foods can lead to tooth destruction as a result of high titratable acidity and a pH of 3.0 or even more acidic6. In literature, the intrinsic factors such as gastro esophageal reflux, vomiting and regurgitation related to gastroesophageal reflux disease, anorexia nervosa and bulimia have been reported to be account for up to 25% of all cases of DE 7. The loss of detailed surface microanatomy, rounded and glazed appearance, hypersensitivity of teeth especially among younger persons are considered as common effects of DE. In extensive cases, DE can compromise aesthetic and performance of the pulp⁵.

Epidemiologic studies with regard to DE have been carried out worldwide. The findings of such studies revealed that the prevalence of DE varies noticeably across the world and between age and gender groups⁶. Dugmore and Rock and Al-Dlaigan et al. identified a significantly higher prevalence of DE in boys than girls^8,9.    In contrast, Bartlett et al.¹⁰ found a higher prevalence of DE in females. Since the number of DE cases is increasing; this could be possibly as a result of variation in dietary habits. The data of DE and its relationship with dietary habits in dental students are scarce, so the purpose of this study was to determine the relationship between dietary habits and DE and prevalence of DE and degree of severity among dental students of LUMHS Jamshoro. The findings of this study will be beneficial for developing the future policies against the various factors in preventing the dental erosion and promoting healthy diet.

METHODOLOGY

Two hundred dental students fulfilling the inclusion criteria were selected from July 2011 to December 2011 with a convenient sampling technique. The design of the study was cross sectional. The dental students with age from 18-25 years of either gender were included and those with gross dental defects such as amelogenesis imperfecta, dentinogenesis imperfecta, rampant caries, fixed orthodontic appliances and medically compromised conditions were excluded. Informed written consent was obtained from the students. All the information regarding variables of study like age, gender and dietary habits were obtained through a pre designed questionnaire ¹¹ consisting of close ended questions. The clinical examination was performed on dental chair using examination instruments: mirror, tweezer and probe. DE and its severity were recorded for anterior and posterior teeth using modified index of Lussi et al ¹². The data were analyzed by SPSS statistical version 16. The variables analyzed were prevalence of dental erosion, its severity and dietary habits. The association between DE and dietary habits was established using chi-square test. P value < 0.05 was taken as significant.

RESULTS

Out of 200 study subjects, 89 (44.5 %) were males and 111 (55.5%) were females. In this study prevalence of DE was found 32 % in study participants (Table I).

Table I: Prevalence of Dental erosion

According to Modified Lussi Index severity of DE was categorized into four groups from normal to severe DE in anterior and posterior teeth respectively. Total 37 subjects (58%) had DE in anterior teeth only, all those subjects had mild DE (Grade 1) . However 08 subjects (12%) were seen with DE in posterior teeth only with Grade 1 DE .  In addition total 19 subjects (30% ) were found to have DE in  both anterior as well as posterior teeth  (Table II,III and IV ). Prevalence of DE was more common in anterior teeth compared to posterior teeth

Table II: Dental Erosion Severity in Anterior Teeth only

Table III: Dental Erosion Severity in Posterior Teeth only

Table IV: Dental Erosion Severity in Anterior and Posterior teeth both

(p=0.000). Out of 64 study subjects with DE 61 (95 %) had mild DE, only 3 (5%) subjects had moderate DE, none of study subjects had severe DE. The frequencies of drinks and foods intake were divided into three categories; once a week, more than once a week or no consumption. The majority of students i.e. 58 (5%) consumed carbonated drinks more than once a week, however intake of lemon tea and sports drinks was rare; total 75% and 56 % of students reported no intake of sport drinks and lemon tea respectively. Consumption of fruits and fruit juices was common in study participants. Total 72 % of students consumed fruits more than once a week and 70 % of students took fruit juices more than once a week. Use of lemon tea and acidic drinks was not frequent. Consumption of drinks through straw was reported by 68 % of students whereas 32% did not use straw. No statistically significant association was observed between DE and various dietary habits such as intake of carbonated drinks, sport drinks, acidic drinks (Table V).

DISCUSSION

In the current study, the prevalence of DE was found 32%. This prevalence is comparable with the results of Caglar et al. ¹³ and Correr et al. ¹⁴  who identified 28% of 11-year-old children and 26% of 12-year-old children affected by DE, respectively. On the other hand, recent surveys highlighted that the prevalence of DE ranging from 11.6 to 100% on the permanent dentition of children in different countries^9,15.  The variation in prevalence among these studies may be in part  elucidated by differences in the diagnostic criteria and indices. Furthermore, outcome of prevalence data may be influenced by socio-economic, cultural, and geographic aspects.

The severity of DE in our study demonstrated that the loss of enamel contour (Grade 1) occurred most frequently (95 %) in anterior and posterior teeth, and

Table V: Frequency of consumption of drinks

only a small proportion of tooth surfaces were affected with dentine exposure (Grade 2) (5%) in anterior as well as posterior teeth, this is in accordance with results of Wang et al. and Peres et al ^11,16.  None of the 64  students with DE  presented with Grade 3 DE which supports the results of Jensdottir et al ¹².

The consumption of fruits and fruit juices was common in students. Total 72 % of students consumed fruits more than once a week and 70 % of students took fruit juices more than once a week. However, this study could not find a significant association between symptoms of DE and frequency of consumption of fruit and fruit juices. Some researchers have also reported no relationship between DE and consumption of fruit as well as fruit juices¹⁷. On contrary, others have found a significant association¹⁹. In case control studies, a substantial risk of DE was observed when citrus foods were consumed  more than twice a day²⁰.

Fruit juices are more likely to cause DE compared to fruit itself ²¹. Conversely, we did not find any significant association between fruit juices and DE (p=0.225). This may be possibly due to low consumption of fruit juice and acidic foods in study participants. This study observed that there was no association between DE and frequency and amount of acidic drinks consumption. DE is more often reported to be related with acidic drinks in children²², adolescents,²³ and adults²⁴ when the utilization was high. However, such association did not occur in children²⁵ when the consumption of acidic drinks was low. In this study, the number of subjects with frequent consumption of acidic drink was low, thereby resulted in a lack of significant association with DE occurrence. An additional possible reason is that current study used cross-sectional design which only

assessed dietary pattern for past two to three months. Dietary patterns during the data collection may not be the same with the dietary pattern when DE occurred. Moreover, DE is a progressive disease which results from frequent and long-term exposure to acidic drinks. This study did not specify whether acidic drinks were consumed with meal or as snack. It has been recommended that acidic food and drinks may be consumed with meals to reduce the risk of tooth erosion as saliva flow is high during meal time²⁶. Finally, risk of DE is a multi-factorial in nature which is also influenced by the tooth composition, structure, saliva composition and milk intake  which were not examined in this study due to time limitation. Further studies are highly recommended to get further insight into the above-said variables.

Limitations Of Study

This study was conducted in the specific geographic area of only one University, not the whole country. It was a single operator based study so operator bias could not be eliminated. There were no data on all potential causes of DE like gastro esophageal reflux disease (GERD).  However, it has been tried in the current study to provide some information about the prevalence of DE and its association with dietary habits.

CONCLUSION

Our study provides evidence that DE is becoming a significant problem in adult University students. DE should receive more attention that promotes awareness in dentists to make an early diagnosis and to evaluate the different etiologic factors that identify children and adults at risk in Pakistan.

REFERENCES

1. Bargen J, Austin L. Decalcification of teeth as a result of obstipation with long continued vomiting. J Am Dent Assoc 1937; 24:1271-1273.
2. Hunter ME,West NX, Hughes JA, Newcombe RG, Addy M. Erosion of deciduous and permanent dental hard tissue in the oral environment. J Dent 2000;28:257263.
3. West NX, Hughes JA, Addy M. Erosion of dentine and enamel in vitro by dietary acids: The effect of temperature, acid character,concentration and exposure time. J Oral Rehabil 2000;27:875-880.
4. Brunton PA,Hussain A. The erosive effect of herbal tea on dental enamel. J Dent 2001;29:517-520.
5. Sushma Shankar Nayak et al. Dental Erosion among 12 Year Old School Children in Belgaum City- A Cross Sectional Study. Pak Paed J 2009;33: 48-57.
6. Jarvinen V, Rytomaa I, Meurman JH . Localisation of dental erosion in referred population. Caries Res ,1992; 26:391-396.
7. Donachie MA,Walls AWG. The tooth wear index: A flawed epidemiological tool in an ageing population group.Community Dent Oral Epidemiol 1996;24:152158.
8. Dugmore CR, Rock WP. The prevalence of tooth erosion in 12-year-old children. Br Dent J. 2004 ;196: 279-282.
9. Al-Dlaigan YH, Shaw L, Smith A. Dental erosion in a group of British 14-year-old, school children. Part I: Prevalence and influence of differing socioeconomic backgrounds. Br Dent J. 2001;190:145-149.
10. Bartlett DW, Coward PY, Nikkah C, Wilson RF. The prevalence of tooth wear in a cluster sample of adolescent schoolchildren and its relationship swith potential Explanatory factors. Br Dent J. 1998;184: 125-129.
11. Wang P, Lin H, Chen J, Liang H: The prevalence of dental erosion and associated risk factors in 12-13-yearold school children in Southern China BMC Public Health 2010;10:478. doi:  1186/1471-2458-10-478
12. Jensdottir  T, Arnadottir B, Thorsdottir I,  Bardow A, Gudmundsson K, Theodors A ,Holbrook P: Relationship between dental erosion, soft drink consumption, and gastroesophageal reflux among Icelanders Clin Oral Invest 2004;8:91-96
13. Caglar E, Kargul B, Tanboga I, Lussi A: Dental erosion among children in an Istanbul public school. J Dent Child (Chic) 2005;72:5-9.
14. Correr GM, Alonso RC, Correa MA, Campos EA, Baratto-Filho F, Puppin-Rontani RM: Influence of diet and salivary characteristics on the prevalence of dental erosion among 12-year-old school children. J Dent Child (Chic) 2009;76:181-187.
15. Ganss C, Klimek J, Giese K: Dental erosion in children and adolescents-a cross-sectional and longitudinal investigation using study models. Community Dent Oral Epidemiol 2001;29:264-271.
16. Peres KG, Armenio MF, Peres MA, Traebert J, De Lacerda JT: Dental erosion in 12-year-old school children: a cross-sectional study in Southern Brazil. Int J Paediatr Dent 2005;15:249-255.
17. Van Rijkom HM, Truin GJ, Frencken JE, Konig KG, van ‘t Hof MA, Bronkhorst EM, Roeters FJ: Prevalence, distribution and background variables of smoothbordered tooth wear in teenagers in the hague, the
    Netherlands. Caries Res 2002;36:147-154.
18. Al-Dlaigan YH, Shaw L, Smith A: Dental erosion in a group of British 14- year-old school children Part II: Influence of dietary intake. Br Dent J 2001;190:258261.
19. Milosevic A, Bardsley PF, Taylor S: Epidemiological studies of tooth wear and dental erosion in 14-year old children in North West England Part 2: The association of diet and habits. Br Dent J 2004;197:479-483.
20. Kunzel W, Cruz MS, Fischer T. Dental erosion in Cuban children associated with excessive consumption of oranges. Eur J Oral Sci. 2000;108:104-9.
21. Grobler SR, Senekal PJ, Kotze TJ. The degree ofenamel erosion by five different kinds of fruit. Clin Prev Dent. 1989;11:23-28.
22. O’Sullivan EA, Curzon ME. A comparison of acidicdietary factors in children with and without dental erosion. ASDC J Dent Child. 2000;67:186-192.
23. Harding MA, Whelton H, O’Mullane DM, CroninM. Dental erosion in 5-year-old Irish school children and associated factors: a pilot study. Community Dent Health. 2003;20:165-170.
24. Johansson AK, Lingstrom P, Birkhed D.Comparison of factors potentially related to the occurrence of dental erosion in high- and low-erosion groups. Eur J Oral Sci. 2002;110:204-211.
25. Luo Y, Zeng XJ, Du MQ, Bedi R. The prevalenceof dental erosion in preschool children in J Dent. 2005;33:115-121.
26. Moynihan PJ. The role of diet and nutrition in theetiology and prevention of oral diseases. Bull World Health Organ. 2005;83:694-699.
    

    ---

    1. PhD Scholar, Dental Materials, Medical Research Centre, LUMHS, Jamshoro.
    2. Associate Professor & Chairman, Department of Operative Dentistry, LUMHS, Jamshoro.
    3. Assistant Professor & Incharge, Department of Science of Dental Materials, LUMHS, Jamshoro.
    4. PhD Scholar, Community Dentistry, Medical Research Centre, LUMHS, Jamshoro.
    5. Assistant Professor, Department of Operative Dentistry, LUMHS, Jamshoro.
    Corresponding author: “Dr. Shahid Ali Mirani” < shahidmirani@hotmail.com >

^{Haroon Rashid1}                                BDS, MDSC  (UK)

The Confocal Laser Scanning Microscope (CLSM) is a type of microscope which utilizes its electronic system for image processing whilst using a laser as the source of light.  The optical image sections it obtains are of superior resolution. CLSM focuses on a single plane and removes the interference caused by the light arriving from the different optical fields across the thickness of the sample. As the images gathered using the microscope are digital images, higher magnifications for optical microscopy can be obtained using the software¹.

The images obtained using CLSM are of higher magnification as compared to the images obtained using the conventional optical microscopy. These images have volumetric and texture details and it is not possible to obtain such details with the conventional ones. The microscope also allows the study of the specimens using transmitted or reflected light meaning that those samples which are not transparent may also be analyzed. Since confocal microscope has the ability to create sharp optical sections, the data gathered from a series of optical sections at short and regular intervals along the optical axis are also used to create a true 3D reconstruction. This is made possible using software which combines the 2D images to create a 3D rendition ².

Confocal microscopy has been widely used in medical and dental research and also for the clinical treatment of various diseases. Along with its research applications in cancer and Alzheimer’s disease, the technique is also commonly used in ophthalmology^3,4, angiogenesis⁵, gynecology⁶, and gastro-intestinal systems⁷. CLSM is routinely used in dentistry to analyze surface roughness, analysis of dental erosion, measuring the actual profile, the numerical roughness parameters and in the studies of micro-tensile bond strength ^8,9,10,11. It has been widely used for studying the surface topography and formation of bio film on dental implants and dental hard tissues ^9,12. Once the data stack has been obtained in the CLSM a non-tactile, non-destructive measurement of the surface roughness is possible. It is an invaluable tool of measurement in dentistry studies. It is also important to mention that confocal microscope maintains the samples under humid conditions and thus avoids the artifacts that may take place with the use of SEM drying specimen preparation techniques. It offers improved relationship between the signal and the noise.

Confocal microscopy, like other devices has some limitations particularly the resolution and the pinhole size ^13,14. It has inherent resolution limitations due to diffraction and the airy disk used in CLSM can limit the maximum resolution which can be attained using the device. The pinhole size, once decreased in CLSM leads to reduced signal to noise ratio and thus more fluorescence from the specimen may be required.

CLSM is widely used in Europe and North America and is one of the most expensive types of microscope available in the market. The microscope is currently being employed for detailed microscopic examination of the cornea in some hospitals across Pakistan. National institute of biotechnology and genetic engineering uses CLSM for research purposes. It is a valuable tool in dental research particularly for accessing the surface roughness of dental biomaterials and the formation of biofilm on dental tissues. However, in Pakistan it is not yet employed in dental research partly because of its cost and partly because of lack of knowledge. Irrespective of financial constraints, it is important that this wonderful device is made available to the local researcher to claim its place in contemporary literature.

REFERENCES

1. Minsky, M. Memoir on inventing the confocal microscope. Scanning 1988;10:128-138.
2. Wilson, T.; Carlini, A.R. Three dimensional imaging in confocal imaging systems with finite-sized detectors. J. Microsc. 1988;141:51-66.
3. Jalbert I, Stapleton F, Papas E, et al. In vivo confocal microscopy of the human cornea. Br J Ophthalmol 2003;87:225-236.
4. Muller LJ, Marfurt CF, Kruse F, et al. Corneal nerves: structure, contents and function. Exp Eye Res 2003; 76:521-542.
5. McDonald DM, Choyke PL. Imaging of angiogenesis: from microscope to clinic. Nat Med 2003;9:713-725.
6. Drezek RA, Richards-Kortum R, Brewer MA, et al. Optical imaging of the cervix. Cancer 2003;98:20152027.
7. Vanden Berghe P, Bisschops R, Tack J. Imaging of neuronal activity in the gut. Curr Opin Pharmacol 2001;1:563-567.
8. Mazzitelli C, Ferrari M, Toledano M, Osorio E, Monticelli F, Osorio R. Surface roughness analysis of fiber post conditioning processes. J Dent Res 2008;87:186-190.
9. Hallgren C, Reimers H, Gold J, Wennerberg A. The importance of surface texture for bone integration of screw shaped implants: an in vivo study of implants patterned by photolithography. J Biomed Mater Res 2001;57: 485-496.
10. Pohl M, Stella J. Quantitative CLSM roughness study on early cavitation-erosion damage. Wear 2002;252:501511.
11. Mannocci F, Sherriff M, Ferrari M, Watson TF. Microtensile bond strength and confocal microscopy of dental adhesives bonded to root canal dentin. Am J Dent. 2001;14:200-4.
12. Dige I, Nilsson H, Kilian M, Nyvad B. In situ identification of streptococci and other bacteria in initial dental biofilm by confocal laser scanning microscopy and fluorescence in situ hybridization. Eur J Oral Sci. 2007;115:459-67.
13. Inoue S, Spring K.R. Microscope image formation. In Video Microscopy the Fundamentals; Plenum Press: New York, 1997;13-118.
14. Wilson, T. The role of the pinhole in confocal imaging system. In Handbook of Biological Confocal Microscopy, 2nd Ed.; Pawley, J.B., Ed.; Plenum Press: New York, 1995;167-182.

1. Assistant Professor, Department of Prosthodontics, Ziauddin College of Dentistry, Ziauddin University, Karachi, Pakistan.

Corresponding author: “Dr. Haroon Rashid” < drh.rashid@hotmail.com >

Tel: : +92-323-3241889