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BASIC AND CLINICAL RESEARCH

Effect of Air-Powder System on Titanium Surface on Fibroblast Adhesion and Morphology

Awad Shibli, Jamil DDS, MS*; Gonzales Silverio, Karina DDS, MS**; Compagnoni Martins, Marilia DDS, MS**; Marcantonio, Elcio Jr., DDS, MS, PhD**; Rossa, Carlos Jr., DDS, MS, PhD**

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doi: 10.1097/01.ID.0000042506.95943.BC
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Abstract

A direct contact between the dental implant surface and surrounding bone is preferred for the long-term success of dental implants. Nevertheless, in spite of a satisfactory osseointegration, this clinical success of dental implants can be guaranteed only if the integrity of periimplant mucosa is maintained by an attachment by hemidesmosomal connection of soft-tissue to the transmucosal implant surfaces. 1,2

On the other hand, it must be emphasized that clinical studies of the interface between gingival tissues and dental implants in humans are hampered by many difficulties, including ethical considerations. Many uncontrollable factors in the oral environment, as well as technical problems on sample preparations, impair experimental studies on soft-tissue behavior. In vitro experiments appear to circumvent most of these difficulties and thus can provide useful information on this subject. 3

Common clinical procedures such as professional maintenance performed with stainless steel and plastic curettes or abrasive pumice or air-powder abrasive system could to lead to alterations on the surface of titanium abutment, impairing, for example, adhesion of fibroblasts to this surface. Comparative experiments on the attachment and growth of human gingival fibroblasts and epithelial cells on titanium with different surface textures were carried out. 4,5 These studies showed that epithelial cells present more extensive migration on rough surfaces. However, gingival fibroblasts showed a more marked and oriented development on porous surfaces, which was also observed by other authors. 6,7

Even though rough surfaces could enhance fibroblast responses, they can also be considered rather disadvantageous because of the possibility of promoting growth and organization of bacterial biofilms, thus facilitating periimplant tissue infections such as mucositis and periimplantitis. 8

The purpose of this in vitro study was to evaluate the effect of using an air-powder abrasive system on titanium abutments on adhesion and morphology of fibroblasts.

Materials and Methods

Cell Lineage

A continuous cell lineage of fibroblastic morphology (McCoy) from the Adolfo Lutz Institute, Sao Paulo, Brazil, was used. These cells were cultured in 25 cm2 flasks with minimum essential media supplemented with 7.5% of fetal bovine serum and 40 μg/mL of gentamicin. The cells were maintained in an incubator at 37°C and 98% humidity atmosphere. Cell suspensions were prepared at the exponential growth phase always from the same passage throughout the experiment. The same batch of supplemented culture media was used throughout the experiment to minimize possible variations on cell growth.

Treatment of Specimens

Twenty-six new commercially pure, titanium healing abutment surfaces (4 mm × 8 mm) (Sterngold; ImplaMed, Attleboro, MA) were used in this study. These abutments were removed from the original packing, cleaned on an ultrasonic device for 10 minutes, and then sterilized by steam heat (autoclave). Care was taken not to contact the abutment cylinder surface with any foreign object other than the test instruments and materials.

Two titanium abutments were designed as negative control (no treatment with air-abrasive system and no cells) and two positive controls (air-powder system treatment and no cells). The remaining 22 specimens were assigned to two experimental groups: control group (no air-powder treatment) and test group (air-powder system) (Prophy-Ceramic II; Dabi Atlante, Ribeirão Preto, SP, Brazil) for 30 seconds on a 45° incidence. The air-powder system was performed with sodium bicarbonate. A single operator used the Prophy-Jet device loaded with sodium bicarbonate on all titanium abutments of the test group. Immediately after treatment, the specimens were coded and individually placed in 24-well plates. To each well, 2 mL of supplemented cell culture media and 1 mL of a cell suspension containing 2 × 105 cells/mL were added. These plates were incubated for 24 hours at 37°C and 98% humidity.

Preparation of Specimens for Scanning Electronic Microscopy

After the incubation period, the culture medium was removed by aspiration from the wells, and the titanium healing abutments were immersed on 2.5% glutaraldehyde for 15 minutes to fix the cells. Following fixation, the specimens were dehydrated in increasing concentrations of ethanol (10, 30, 50, 70, 90 and 100%) and placed in a vacuum dissecator where they remained during 5 days. The healing abutments were then mounted on metallic stubs and coated with 20 nm of gold to be observed and photographed in the scanning electronic microscope at ×500 and ×1000 for density and morphology evaluations, respectively. The assistant microscopy technician, who was unaware of the coding that identified the experimental groups randomly, determined the photographic fields.

Cell Morphology Assessment

The photomicrographs were submitted to three independent and previously calibrated examiners (examiner 1 × 2, kappa: 1.00; examiner 1 × 3, kappa 0.84; examiner 2 × 3, kappa 0.84) who evaluated cell morphology according to an index system proposed by Gamal et al 9 and modified by us. 10 Briefly, the scoring system was as follows: score 0, no cells present; score 1, only flattened cells; score 2, only rounded cells; and score 3, presence of both rounded and flattened cells.

Cell counting was performed on all photomicrographs using a black paper mask in which a window of 3 cm2 (corresponding to a “real” area of approximately 200 um2) was cut. This mask was superimposed on the photomicrographsm, and triplicate counts of the number of the cells adherent to the titanium abutments were made for each group. A single examiner, blind to experimental groups coding, performed these counts.

Data Analysis

Experimental groups were considered independent, and data related to the number of cells, although discrete in nature, were considered to present an approximately normal distribution. Comparison between the mean number of cells present on three random fields (200 um2 area/each) was performed with a nonpaired t test. Because cell morphology was assessed by an index system (scores ranging from 0–3), a nonparametric Mann-Whitney test was used to compare the mean ranks of the experimental groups. The null hypothesis for both experiments (cell number and morphology) was that there was no difference between the experimental groups. Significance level was always set to 95%.

Results

The distribution of morphology scores (Fig. 1) according to experimental group Mann-Whitney tests did not indicate significant differences between groups (P > 0.05), suggesting that cell morphology was not affected by treating the healing abutments with the air-abrasive system.

Fig. 1
Fig. 1:
Frequency distribution of the percentage of scores for cell morphology according to the experimental groups.

The nonpaired t test indicated that the number of fibroblasts was significantly different (P = 0.001) between groups. The control group (Figs. 2 and 3) presented a significantly greater amount of cells (71.44 ± 31.93) in comparison with the test group (35.31 ± 28.14) (Figs. 4 to 6).

Fig. 2
Fig. 2:
Scanning electron microphotograph of negative control titanium abutment (×1000 original magnification).Fig. 3. Scanning electron microphotograph of control group (×1000 original magnification). Fig. 4. canning electron microphotograph of control titanium abutment after air-powder treatment (×500 original magnification). Fig. 5. Scanning electron microphotograph of control group abutment presenting fibroblasts on its surface (×1000 original magnification).
Fig. 6
Fig. 6:
Mean and standard deviation of number of cells according to experimental groups.

Discussion

Acquisition and maintenance of an effective attachment around the cervical portion of a dental implant is essential to establish a favorable prognosis. The periimplant seal provides a biologic barrier between the oral environment and periimplant bone tissue. The disruption of this seal by inflammatory periimplant disease can permit increased accessibility of biofilm-derived substances into the connective tissues. Several studies have tested various measures for cleaning smooth implant surfaces. 8,11–13 Surface cleaning with an air-powder abrasive system has been suggested. 14–16

The results of this in vitro study showed that proliferation and migration fibroblasts are possible on titanium surfaces, in accordance with Gould et al. 17, whose in vitro results indicated a hemidesmosomal connection between epithelial cells and titanium surfaces. It was also shown that even the orientation of fibroblasts could be influenced by titanium surface characteristics. 4,18

Using an air-powder abrasive system with sodium bicarbonate for 30 seconds on a 45° device on commercially pure titanium did not alter the morphology of fibroblasts. In agreement with the literature, 19,20 morphology of these cells was predominantly elongated or flattened (bipolar or multipolar), which was considered a sign of adhesion to the substrate. This indicates that surface roughness and the presence of particles of bicarbonate were not able to alter this phenotypic expression of fibroblasts. However, what consequences this type of surface instrumentation can have on the attachment of periimplant soft tissues in the long-term remains unclear.

On the other hand, on titanium surfaces treated with an air-powder abrasive system, a significant decrease in the number of fibroblasts was observed in comparison with nontreated control titanium surfaces. Another study has documented similar results after surface treatment with stainless steel curettes. 21 There are some in vitro data suggesting that smooth surfaces are superior in promoting fibroblast proliferation as well as the number of cells attaching to the surface. 22 These results can be attributed to the release of toxic ions from the titanium alloy 23 or to the presence of powder particles on instrumented surfaces, which can disturb cellular adhesion.

Results obtained in these studies show that the nature and surface geometry of the implant surfaces may influence gingival fibroblasts attachment in vivo, in agreement with our data. However, one has to bear in mind the limitation of the methods used in this study when considering these results. In this study, a continuous lineage of fibroblastic cells was used. The advantages of this type of culture are the rapid proliferation of cells (reducing the probability of contamination), the infinite life-span of cells, allowing for many repetitions of experiments, in addition to the fact these cells are also easier to grow and maintain. In consideration of the purpose of this study, which was to perform an initial evaluation of adhesion and proliferation of fibroblasts on titanium surfaces after treatment with an air-powder system, cells from continuous lineages are considered adequate. 24

Appropriate care was taken to minimize possible sources of variation on the assay. This care included preparation of cell suspensions at the exponential growth phase as well as always obtaining these cells from the same 75 cm2 cell culture flask to avoid possible differences on cell behavior caused by variations in culture conditions. In this sense, the same supplemented culture medium batch was used throughout the study.

Conclusion

Treatment of titanium abutments with an air-powder device using sodium bicarbonate significantly reduced the number of fibroblasts attached to these surfaces. On the other hand, no morphologic alterations were observed on the cells present on treated titanium surfaces, indicating that the adhesion of fibroblasts was not significantly affected. Clinically, these findings indicate that using an air-powder abrasive system on titanium abutments to remove bacterial biofilm during treatment of periimplant mucositis or maintenance care does not reduce the biocompatibility of these surfaces.

References

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Abstract Translations [German, Spanish, Portuguese, Japanese]

AUTOR(EN): Jamil Awad Shibli, DDS, MS*, Karina Gonzales Silverio, DDS, MS**, Marilia Compagnoni Martins, DDS, MS***, Elcio Marcantonio Jr., DDS, MS, PhD****, Carlos Rossa Jr., DDS, MS, PhD*****. *Abteilung für Orthodontie, zahnmedizinische Fakultät in Araraquara - staatliche Universität Sao Paulo (UNESP) - Araraquara, Sao Paulo, Brasilien, Abteilung für Oralbiologie, dentalmedizinische Fakultät, staatliche Universität von New York in Buffalo, Buffalo, NY. **Abteilung für Orthodontie, zahnmedizinische Fakultät in Araraquara - staatliche Universität Sao Paulo (UNESP) - Araraquara, Sao Paulo, Brasilien. ***Abteilung für Orthodontie, zahnmedizinische Fakultät in Araraquara - staatliche Universität Sao Paulo (UNESP) - Araraquara, Sao Paulo, Brasilien. ****Abteilung für Orthodontie, zahnmedizinische Fakultät in Araraquara - staatliche Universität Sao Paulo (UNESP) - Araraquara, Sao Paulo, Brasilien. *****Abteilung für Orthodontie, zahnmedizinische Fakultät in Araraquara - staatliche Universität Sao Paulo (UNESP) - Araraquara, Sao Paulo, Brasilien. Schriftverkehr:Carlos Rossa Jr., DDS, Departamento de Periodontia, Faculdade de Odontologia de Araraquara, UNESP, Rua Humaita, 1680, 14801 - 903, Araraquara, Sao Paulo, Brasilien. Fax: +55 16 201 – 6314; eMail: crossajr@foar.unesp.br

ZUSSAMENFASSUNG:Zielsetzung: Innerhalb vorliegender Studie sollte eine Auswertung bezüglich der Anzahl und Morphologie von auf der Oberfläche von maschinell bearbeiteten, zur Heilung eingesetzten Titanstützzähnen wachsenden Fibroblasten erfolgen, die mit einem speziellen Druckluftsystem behandelt wurden. Materialien und Methoden: Es erfolgte eine Aufteilung von insgesamt 26 Stützzähnen auf zwei Versuchsgruppen: die Zähne der ersten Gruppe blieben als Kontrollmedien unbehandelt, die der zweiten wurden für 30 Sekunden mit dem Prophy-Jet Druckluft ausgesetzt. Die Präparate wurden zusammen mit Fibroblastzellen auf Mikrotiterplatten aufgebracht und für 24 Stunden in den Brutofen gestellt. Nach Ablauf dieser Zeit wurden sie im Labor routinemäßig auf die Rasterelektronenmikroskopie vorbereitet. Die Untersuchung der Präparate und die Ermittlung der Zellanzahl erfolgten mittels Abtastung (Auflösung 350X) eines Bereiches von ca. 200 μm2. Ergebnisse: Die morphologischen Vergleichswerte beider Gruppen stimmten weitestgehend überein (p>0,05), allerdings fanden sich bei der Kontrollgruppe wesentlich mehr Zellen (71,44 ± 31,93, Mittelwert ± s.d.) als bei der Gruppe mit den behandelten Implantaten (3531 ± 28,14). Diese Werte wurden durch Einzeltest ermittelt. Schlussfolgerung: Die Behandlung der Oberflächen der für die Heilungsphase eingesetzten Titan-Stützzähne mit einem luftgestrahlten Prophylaxesystem wirkte sich verringernd auf die Zellvermehrung aus, ohne die Zellmorphologie zu beeinflussen.

SCHLÜSSELWÖRTER: Zahnimplantate, Zellkultur, Titan, Fibroblasten, Erhaltung, Rasterelektronenmikroskop

AUTOR(ES): Jamil Awad Shibli, DDS, MS*, Karina Gonzales Silverio, DDS, MS**, Marilia Compagnoni Martins, DDS, MS***, Elcio Marcantonio Jr., DDS, MS, PhD****, Carlos Rossa, Jr., DDS, MS, PhD*****. *Departamento de Periodontología, Facultad de Odontología en Araraquara - Universidad Estatal de San Pablo (UNESP), Araraquara, SP, Brasil, Departamento de Biología Oral, Facultad de Medicina Oral, Universidad Estatal de Nueva York en Buffalo, Buffalo, NY. **Departamento de Periodontología, Facultad de Odontología en Araraquara - Universidad Estatal de San Pablo (UNESP), Araraquara, SP, Brasil. ***Departamento de Periodontología, Facultad de Odontología en Araraquara - Universidad Estatal de San Pablo (UNESP), Araraquara, SP, Brasil. ****Departamento de Periodontología, Facultad de Odontología en Araraquara - Universidad Estatal de San Pablo (UNESP), Araraquara, SP, Brasil. *****Departamento de Periodontología, Facultad de Odontología en Araraquara - Universidad Estatal de San Pablo (UNESP), Araraquara, SP, Brasil. Correspondencia a: Carlos Rossa Jr., DDS, Departamento de Periodontia, Faculdade de Odontologia de Araraquara - UNESP, Rua Humaita 1680, 14801-903 Araraquara, SP – Brasil. Fax: 55 16 201-6314; Correo electrónico: crossajr@foar.unesp.br

ABSTRACTO: Propósito: Evaluar el número y morfología de los fibroblastos que han crecido en los postes de curación de titanio pulidos a máquina tratados con un sistema de polvo de aire. Materiales y Métodos: Veintiséis postes fueron asignados a 2 grupos experimentales: control (sin tratamiento) y tratados - expuestos a Prophy-Jet durante 30 segundos. Los especímenes fueron incubados durante 24 horas con células fibroblásticas en placas con múltiples pocillos, seguidos por procesamiento de rutina en laboratorios para el análisis SEM. Los especímenes fueron fotografiados en 350X y el número de células se contaron en una área de aproximadamente 200 um2. Resultados: No se encontraron diferencias significativas en morfología entre los grupos (p > 0,05), sin embargo, el grupo de control presentó una cantidad más importante de células (71,44 ± 31,93, mediana ± d.s.) en comparación con el grupo tratado (35,31 ± 28,14), según lo indica una prueba T sin pares (p = 0,001). Conclusión: El uso de un sistema de profilaxis abrasivo de aire sobre la superficie de los postes de curación de titanio redujo la proliferación de células pero no tuvo influencia en la morfología de las células.

PALABRAS CLAVES: Implantes dentales, cultivo de células, titanio, fibroblastos, mantenimiento, microscopía por escaneado de electrones

AUTOR(ES): Jamil Awad Shibli, DDS, MS*, Karina Gonzales Silverio, DDS, MS**, Marilia Compagnoni Martins, DDS, MS***, Elcio Marcantonio Jr. DDS, MS, PhD****, Carlos Rossa Jr., DDS, MS, PhD*****. *Departamento de Periodontologia, Faculdade de Odontologia de Araraquara – Universidade Estadual de São Paulo (UNESP) – Araraquara, SP, Brasil. Departamento de Biologia Oral, Faculdade de Medicina Odontológica, Universidade do estado de Nova York, Búfalo, Búfalo, NY. **Departamento de Periodontologia, Faculdade de Odontologia de Araraquara – Universidade Estadual de São Paulo (UNESP) – Araraquara, SP, Brasil. ***Departamento de Periodontologia, Faculdade de Odontologia de Araraquara – Universidade Estadual de São Paulo (UNESP) – Araraquara, SP, Brasil. ****Departamento de Periodontologia, Faculdade de Odontologia de Araraquara – Universidade Estadual de São Paulo (UNESP) – Araraquara, SP, Brasil. *****Departamento de Periodontologia, Faculdade de Odontologia de Araraquara – Universidade Estadual de São Paulo (UNESP) – Araraquara, SP, Brasil.Correspondências devem ser enviadas a: Carlos Rossa Jr., DDS, Departamento de Periodontologia, Faculdade de Odontologia de Araraquara – UNESP, Rua Humaita, 1680, 14801-903 Araraquara, SP – Brasil. Fax: +55 16 201-6314, Email: crossajr@foar.unesp.br

SINOPSE:OBJETIVO: avaliação do número e morfologia dos fibroblastos desenvolvidos em pivôs de cicatrização de titânio usinado, tratados com um sistema de jato abrasivo. MATERIAIS E MÉTODOS: vinte e seis pivôs foram distribuídos em dois grupos experimentais: controle (sem tratamento) e com tratamento – expostos ao Prophy-Jet por 30 segundos. As espécimes foram incubadas por 24 horas com células fibroblásticas em placas de orifícios múltiplos (multiwell), seguidas de processamento laboratorial rotineiro para a análise SEM.Os espécimes foram fotografados em 350X e foi realizada a contagem do número de células em uma área de aproximadamente 200 um2. RESULTADOS: não foram encontradas diferenças significativas na morfologia entre os grupos (p>0,05) entretanto, o grupo de controle apresentou uma quantidade de células significativamente maior (71,44 ± 31,93, média ± s.d.) em comparação com o grupo com tratamento (35,31 ± 28,14), conforme indicado por um teste t sem paridade (p=0,001). CONCLUSÃO: a utilização do sistema profilático de jato de areia na superfície dos pivôs de cicatrização de titânio reduziram a proliferação das células mas não influenciaram a morfologia da célula.

PALAVRAS-CHAVES: implantes odontológicos, cultura celular, titânio, fibroblasto, manutenção, microscopia eletrônica de varredura

FIGURE

Figure
Figure
Keywords:

cell culture; titanium; fibroblasts; maintenance; scanning electron microscopy

© 2003 Lippincott Williams & Wilkins, Inc.