Winner 2003 - The IADR.CED Visiting Scholar Stipend

Agata Czajka-Jakubowska

IADR – CED Visiting Scholar Stipend Support Report

            Dr. Clarkson has a split faculty appointment between University of Michigan’s School of Dentistry, U.S.A. and Leeds Dental Institute, U.K. I have therefore visited both of these institutions to learn the research techniques which are being used in our funded project investigating a possible mechanism for the aetiology of dental fluorosis. I will outline the techniques learned at the two institutions during my visits.

Leeds Dental Institute

            I was able to use the experience gained during my Ph.D. (“The effect of the Polypherols in Green Tea on the Caries Experience in Rats”) to obtain the skulls of rats which I had been dosed with fluoride concentrations of 0, 25, 50, 75 ppm. This part of the study was carried out in Poznan, Poland under my direct supervision. At Leeds, under the supervision of Drs. Clarkson and Robinson I removed the mandible from the skull, extracted the central incisor teeth and identified the three developmental enamel zones, secretory, transition and maturation of the incisor teeth. I then dissected these zones and stored the enamel samples for later separation of the individual crystals.

University of Michigan

            Under the supervision of Drs. Clarkson and Chen (a Associate Research Scientist collaborating with Dr. Clarkson) I learned to separate the individual crystals from the enamel samples following a protocol which is outlined later. Having separated the crystals they were subjected to Atomic Force Microscopic (AFM) imaging and analysis of the roughness of the surface of the crystals. I participated in this analysis by distributing the enamel crystals on the mica surfaces prior to imaging in the microscope. I also had hands-on experience of using the microscope but still need more time to master this technique.

Enamel Crystal Isolation

            Individual crystals from maturation-stage enamel were obtained from the mandibular incisors of four-week-old male Sprague Dawley rats (Robinson et al., 1974; Hiller et al., 1975). (The animal use protocol was reviewed and approved by the University’s committee on Use and Care of Animals).

All detectable traces of matrix protein were removed from the enamel samples by a sequential extraction procedure described by Robinson et al. (1995). Briefly, enamel particles were first extracted with 0.1 M phosphate buffer, pH 7.4, to desorb mineral-bound proteins and components dissolved in enamel fluid. After centrifugation, ww re-extracted the insoluble pellet by re-suspending it in fresh phosphate buffer. This was repeated a total of 6 times. The pelleted material was then further extracted with the use of 50 mM Tris containing 4 M urea at pH 7.4 to dissolved aggregated precipitated protein. The insoluble residue was then re-extracted for a further 6 times with 0.1 M phosphate buffer, pH 7.4, to ensure final desorption and dissolution of any mineral-bound components. The final residue was washed with distilled water with the pH adjusted to 7.4 so that all traces of buffer and urea would be removed. The crystals were then treated with 3% hypochlorite for oxidation of traces of the organic materials and washed with distilled water. The crystals were finally dispersed in HPLC-grade methanol by sonication.

Atomic Force Microscopy

            The enamel crystals were sonicated for 2 min in HPLC-grade methanol to reduce aggregation. Two 5-µL quantities of this suspension were then pipetted onto freshly cleaved mica. The methanol was evaporated rapidly, leaving a coating of dispersed hydroxyapatite crystals.

            All samples were imaged in tapping mode in air, by means of a Nanoscope IIIa Multimode AFM and controller (Digital Instruments, Santa Barbara, CA, USA) equipped with a 120 µm x 120 µm J-type scanner. Commercially available tapping mode cantilevers, TESP, were used (Digital Instruments).

            Over the next year I will revisit Leeds Dental Institute to use Ion Chromatography to determine the concentration of fluoride in the enamel crystals. I will also return to Michigan to obtain more hands on experience in using the AFM where I will conduct studies to establish if there is a difference in the binding capacity of enamel proteins to non-fluorotic and flurotic crystals.

            During this Visiting Scholar period Dr. Clarkson has visited Poznan several times where we have successfully dissected the three enamel zones from rat incisors. We are also in the process of isolating the crystals in a laboratory at my home institution.

            This year we have had two abstracts accepted reporting this work one in Poznan, Poland at a National Meeting (Saldent) hosting over 1,000 polish dentists and scientists; and one that will be presented in Amsterdam in September at the IADR-CD meeting. One previous presentation using the fluorotic enamel crystals was made at the IADR/AADR meeting in Baltimore, U.S.A.

            I have also submitted a paper co-authored with my collaborators in Michigan reporting our results on the surface roughness of fluorotic versus non fluorotic crystals to the Journal of Dental Research.

475 Fluoride`s effect on the surface roughness of developing enamel crystals

A. CZAJKA-JAKUBOWSKA¹, H. CHEN², and B.H. CLARKSON², ¹ University of Medical Sciences, Poznañ, MI, Poland, ² University of Michigan, Ann Arbor, USA

It has been shown that the detrimal effects of high doses of fluoride occur during the late transition and early maturation stage of enamel development. It has been proposed that these detrimal effects are caused by the delayed removal of enamel proteins resulting in impairment of enamel crystal growth. We have reported that this delayed removal may be caused because the surface of fluorotic enamel crystals are rougher than non fluorotic crystals and, therefore, bind ameloblastin and amelogenin more tightly. statistically

Objective: To establish whether the surface roughness of fluorotic crystals was dose dependent and whether crystals from both the transition and maturation stages of enamel development are effected in a dose dependent manner.

Methods: Enamel crystals were obtained from developing mandibular incisors of Wistar rats. Litter mates were raised either in the absence of fluoride (control) or with 25, 50, 75 ppm fluoride administered in the drinking water. Both maturation and transition stage enamel were microdisected from the developing incisors. Roughness measurements (RMS) were obtained for the crystals after imaging on a Nanoscope IIIa Multimode Atomic Force microscope (AFM) in tapping mode.

Results: Both maturation and transition stage fluorotic enamel crystals showed statistically increased surface roughness when compared with the non-fluoride controls and the RMS for 75 ppm dose was statistically greater than that for the 25 and 50 ppm dose. No difference was seen between 25 and 50 ppm in both groups. (Maturation stage enamel RMS: control, 0.53±0.18; 25 ppm, 0.65±0.21; 50 ppm, 0.71±0.20; 75 ppm, 0.85±0.28. Transition stage RMS: control, 0.5±0.16; 25 ppm, 0.85±0.27; 50 ppm, 0.86±0.32; 75 ppm, 1.07±0.35).

Conclusion: Surface roughness of fluorotic crystals in both maturation and transition stage enamel depends on the fluoride concentration when administred systemically and increases as the fluoride concentration increases although this effect is not linear.(IADR.CED Visiting Scholar Stipend)

Joint Meeting of the Continental European and Scandinavian (NOF) Divisions of the IADR (September 14 - 17, 2005)

Atomic Force Microscopy (AFM) in dental research.

B.Clarkson*, H.Chen, A.Czajka-Jakubowska

University of Michigan School of Dentistry

Department of Cariology, Restorative Sciences and Endodontics

Klinika Stomatologii Zachowawczej i Periodontologii

Akademii Medycznej im. K. Marcinkowskiego w Poznaniu

The AFM is a laser imaging microscope which allows nanoscale objects to be imaged and their surface properties characterized. The microscope can be operated in either a tapping or continuous mode; and the specimen can either be wet or dry. Simply the AFM laser beam is reflected from a mirror sitting on “top” the surface of a probe which will be either dragged or tapped across the surface of the specimen. Not unlike a blind man using a stick to probe the ground in front of him so that he avoids tripping on a stone, tree root, or uneven surface. Thus the images generated by the AFM are height images. The underlating surface of the specimen deflects the probe, changes the orientation of the laser beam which the microprocessor generates into an image. In dentistry AFM has been used to measure the roughness of surfaces, for example, non fluorotic versus non-fluorotic enamel crystals; to image the binding of proteins to enamel crystals which may effect enamel crystal growth; shape and size of bone, enamel and dentin crystals. Examples of these images will be presented in the poster.

2143 Binding of Amelogenin to Systemically Fluoridated Enamel Crystals

N. SPENCER¹, H. CHEN¹, A. CZAJKA-JAKUBOWSKA², J. SIMMER¹, and B.H. CLARKSON¹, ¹ University of Michigan, Ann Arbor, USA, ² University of Medical Sciences, Poznan, Poland, Ann Arbor, MI, USA

The effectiveness of systemic fluoride as a preventative agent against dental caries is dose dependent. One unfortunate side-effect of systemic fluoride administration is that as the concentration increases above 1 ppm so does the incidence of dental fluorosis. The cellular mechanisms of dental fluorosis are not well understood, but it has been proposed that impaired removal of enamel matrix proteins, from enamel crystal surfaces may impede crystal growth, leading to hypoplastic defects in enamel.

Objective: To describe the binding capacity of systemically fluoridated enamel crystals to amelogenin, an important enamel matrix protein.

Methods: Enamel crystals were obtained from rat mandibular incisors. Litter mate rats were raised either in the absence of fluoride (control), or with 50 ppm fluoride administered in the drinking water from day 21. Crystals were imaged on a Nanoscope IIIa Multimode Atomic Force Microscope (AFM) in fluid tapping mode. The crystals were then added to an aqueous solution containing 100ug/mL amelogenin adjusted to pH 7.4 for 30 minutes. 5uL of the suspension were then pipetted onto a freshly cleaved mica surface. The crystals were then imaged in air using AFM. Roughness measurements (Ra) were obtained for both sets of crystals before and after protein binding.

Results: Roughness measurements showed that fluoridated enamel was significantly rougher than control crystals. The roughness of both crystals significantly increased following protein binding. Roughness measurements were: Control crystals 0.52±0.18, control after amelogenin binding 0.85±0.35, systemically fluoridated crystals 0.71± 0.2, systemically fluoridated crystals after amelogenin binding 1.21±0.37.

Conclusion: The binding of amelogenin to enamel crystals increases their roughness. The greater roughness increase seen in fluoridated crystals may indicate that they bind more protein than control crystals. Further work is being done to determine whether these proteins are also binding with an increased affinity.

IADR/AADR/CADR 83rd General Session (March 9-12, 2005)

3592 Fluoridated Enamel Crystals Bind Ameloblastin More Tightly than Control Crystals

N.J. SPENCER¹, H. CHEN¹, A. CZAJKA-JAKUBOWSKA², and B.H. CLARKSON¹, ¹ University of Michigan, Ann Arbor, USA, ² University of Medical Sciences, Poznan, Poland

The effectiveness of systemic fluoride as a preventative agent against dental caries is dose dependent. One unfortunate side-effect of systemic fluoride administration is that as the concentration increases above 1 ppm so does the incidence of dental fluorosis. The cellular mechanisms of dental fluorosis are not well understood, but it has been proposed that impaired removal of enamel matrix proteins, from enamel crystal surfaces may impede crystal growth, leading to hypoplastic defects in enamel.

Objective: To describe and compare the binding capacity of fluorotic and non-fluorotic enamel crystals for recombinant ameloblastin, an important enamel matrix protein.

Methods: Enamel crystals were obtained from rat mandibular incisors. Litter mate rats were raised either in the absence of fluoride (control), or with 75 ppm fluoride administered in the drinking water from day 21. Crystals were imaged on a Nanoscope IIIa Multimode Atomic Force Microscope (AFM) in fluid tapping mode. The crystals were then exposed to 5 uL of recombinant ameloblastin and immediately imaged. Finally, the crystals were washed in increasing concentrations of phosphate buffer, pH 7.4, and imaged. Roughness measurements (RMS) were obtained for both sets of crystals after each stage.

Results: Roughness measurements of the surface of the crystals confirmed that fluoridated enamel bound ameloblastin more tightly. Control enamel returned to normal roughness after a 100mM phosphate buffer wash (RMS=0.56), while the fluoridated enamel roughness remained high (RMS=0.81). Fluorotic crystal roughness did not return to normal until a 200mM phosphate buffer wash was applied.

Conclusion: Fluorotic enamel crystals have a higher affinity for ameloblastin than the control enamel crystals. Further work is being done to determine whether this increased affinity is due to an increased roughness of the enamel crystal itself, or due to an alteration of the surface charge of the crystal. Research supported by NIH Grant #12899.

IADR/AADR/CADR 82nd General Session (March 10-13, 2004)

Wpływ fluoru na chropowatość powierzchni kryształu szkliwa w różnych fazach jego rozwoju.

A.Czajka-Jakubowska*, H.Chen, B.H.Clarkson

Klinika Stomatologii Zachowawczej i Periodontologii

AM w Poznaniu

University of Michigan School of Dentistry

Department of Cariology, Restorative Sciences and Endodontics

Nadmierna ekspozycja szkliwa na działanie fluoru w końcowym etapie fazy przejściowej i wczesnej fazy dojrzewania tej tkanki jest prawdopodobnie główną przyczyną powstawania zaburzeń pochodzenia rozwojowego, a stopień chropowatości powierzchni szkliwa odgrywa w tym procesie znaczącą rolę. Cel Celem pracy było określenie wpływu różnych stężeń fluoru na topografię powierzchni kryształu szkliwa w fazie przejściowej i dojrzewania. Materiał i metodyka Doświadczenie przeprowadzono na szczurach białych rasy Wistar, które podzielono na 4 grupy. W grupie I zwierzęta pojono wodą dejonizowaną, w grupach II, III i IV roztworami wodnymi o zawartości jonów fluorkowych odpowiednio:

25 ppm, 50 ppm, 75 ppm. Materiał badawczy stanowiły kryształy szkliwa w fazie przejściowej (A) i dojrzewania (B) izolowane z siekaczy szczurów. Pomiar stopnia chropowatości (RMS) uzyskiwano na podstawie obrazowania powierzchni kryształów szkliwa przy użyciu cyfrowego mikroskopu skaningowego sił atomowych ( Nanoskop III a). Wyniki RMS dla kryształów szkliwa w fazie przejściowej: IA 0.5±0.16; IIA 0.85±0.27; IIIA 0.86±0.32; IVA 1.07±0.35. RMS dla kryształów szkliwa w fazie dojrzewania: IB 0.53±0.18; IIB 0.65±0.21; IIIB 0.71±0.20; IVB 0.85±0.28. Statystycznie istotną różnicę stwierdzono we wszystkich grupach badawczych w odniesieniu do grup kontrolnych IA i IB. Wniosek Stopień chropowatości powierzchni kryształów szkliwa w różnych fazach jego rozwoju zależy od stężenia jonów fluorkowych dostarczanych endogennie i wzrasta wraz ze wzrostem stężenia fluoru.