Interactions of fibroblasts with soldered and laser-welded joints

R. Solmi^{, , a}, D. Martini^b, M. Zanarini^c, S. Isaza Penco^d, L. Rimondini^{e, f}, P. Carinci^a, G. Borea^c and A. Ruggeri<sup>b

a</sup> Istituto di Istologia ed Embriologia Generale, Università di Bologna,Via Belmeloro 8, Bologna 40126, Italy
^b Dipartimento di Scienze Anatomiche Umane e Fisiopatologia dell'Apparato Locomotore, Università di Bologna, Bologna, Italy
^c Dipartimento di Scienze Dentali, Università di Bologna, Bologna, Italy
^d Dentaurum Italia, Bologna, Italy
^e Corso di Laurea in Odontoiatria e Protesi Dentaria, Università di Insubria, Como-Varese, Italy
^f Servizio di Chirurgia Sperimentale-Istituti Ortopedici Rizzoli, Bologna, Italy

Received 21 February 2003;  accepted 14 July 2003. ; Available online 6 September 2003.
Biomaterials
Volume 25, Issue 4 , February 2004, Pages 735-740

1. Abstract

Relatively little is known about the biocompatibility of the soldered or laser-welded joints of dental appliances. We investigated the reaction of human gingival fibroblasts cultured in vitro in direct contact with samples of soldered and laser-welded joints from orthodontic lingual arches. Contrast phase light microscopy was used to evaluate cell adhesion, morphology and proliferation after 6 and 24 h and after 7 and 16 days. Scanning electron microscopy (SEM) was performed at 16 days.

Our in vitro findings provide evidence that laser-welded orthodontic appliances have superior fibroblast biocompatibility.

Author Keywords: Soldering; Welding; Orthodontic joints; Human gingival fibroblasts; SEM

2. Article Outline

1. Introduction

2. Materials and methods

2.1. Materials

2.2. Cell culture

2.3. Adhesion and proliferation analysis

2.4. Scanning electron microscopy (SEM)

2.5. Statistical analysis

3. Results

4. Discussion

5. Conclusion

Acknowledgements

References

3. 1. Introduction

Orthodontic manufacturers currently use laser-welding to produce highly resistant components (e.g. expanding screws, brackets with mesh, laser-welded structured bases and extra-oral arches) for distribution to dental laboratories and orthodontic warehouses [1]. However, soldering techniques are still frequently used by small-scale orthodontic laboratories for more personalised items such as components of Delaire's and soldered arches. With soldered products, the use of a different alloy as a supply material (alongside that used for welding) can trigger an oxidation process, leading to corrosion of the metal surface and subsequent release of metallic ions. This has caused concern regarding the biocompatibility of soldering [2, 3 and 4]. The metallic ions released in the oral cavity as a consequence of corrosion of conventionally soldered appliances can lead to allergic manifestations and local toxicity. Little is known about the onset of localised cytotoxicity associated with the joints of orthodontic appliances except that its incidence seems to be directly proportional to the release of metallic ions [5 and 6]. In vitro cell-culture studies comparing the biocompatibility of soldered and laser-welded joints are currently lacking.

In the present study, we evaluated the fibroblasts' behaviour in terms of adhesion, morphology and proliferation.

4. 2. Materials and methods

2.1. Materials

Lingual arches were obtained by soldering and/or laser-welding a molar band ("Dentaform Snape", thickness 0.18 mm, stainless steel No. 1.001: Cr 17-19%; Ni 11-13%; Dentaurum, Turnstrab, Ispringen, Germany) with an orthodontic wire ("Remanium", inox steel No. 1.003: Cr 16-18%, Ni 6-9%; Dentaurum). For soldering, small bars made of Ag 5%, Cu 16% and Zn 24% with a melting point of 660°C (Dentaurum) were used as supply material. They were soldered by an electrochemically generated hydrogen-oxygen flame [7]. For laser-welding, a "Laser Desktop" machine (Dentaurum) was used. To permit cell culture, the soldered and laser-welded samples were all autoclaved for 30 min at 121°C.

2.2. Cell culture

An interdental gingival papillae biopsy (2×5 mm²) was obtained from a 30-yr-old healthy donor who had provided informed written consent. The biopsy was divided into 0.2 mm³ pieces and placed in three T-30 flasks (Becton Dickinson Labware, Lincoln Park, NJ, USA) under sterile conditions. The pieces were explanted [8] in Iscove's Modified Dulbecco's Medium (IMDM) (Biowhittaker, Walkersville, MD, USA) supplemented with 10% foetal bovine serum (Life Technologies, Paisley, Scotland, UK), 200 U/ml penicillin and 200 mg/ml streptomycin (Biowhittaker) and incubated at 37°C in a humidified atmosphere containing 5% CO₂. After 2-3 days, epithelial cells expanded from the biopsy fragments. Fibroblasts appeared about 1 week later; because of their migratory capacity, they settled at some distance from the fragments. At confluence (after 20-40 days of primary culture), typically elongated fibroblasts strongly outnumbered the characteristically square-shaped epithelial cells (IMDM is selective for fibroblasts since epithelial cells require additional elements such as bovine pituitary extract, norephinephrine, epidermal growth factor). Trypsinisation was then performed and the cells were expanded from one to two T-30 flasks. Four passages were needed to obtain fibroblastic homogeneity. At the fifth passage, each cell strain was checked for mycoplasma contamination (Biological Industries, Kibbutz Beit-Haemek, Israel). Using 3.5 cm diameter tissue culture dishes (Life Technologies) 10⁵ fibroblasts were then seeded on laser-welded samples (4 dishes), soldered samples (4 dishes) and alone (4 dishes). Medium sterility was controlled in two further dishes. The culture medium was replaced on days 3 and 7.

2.3. Adhesion and proliferation analysis

On the substrate, fibroblast adhesion and proliferation were evaluated by phase-contrast light microscopy (LEITZ Labovert FS Vario-Orthomat 2, Wetzlar, Germany) at 6 and 24 h, and at 7 and 16 days of culture. The studied substrates were the laser welded joints (L) and the soldered joints (S); while the flask surface (C) was considered the control surface. Adhesion was determined by the shape of fibroblasts on the substrate. In normal conditions they are elongated. A round shape indicates unsuccessful adhesion while a flat, non-elongated shape provides evidence of inadequate adhesion. Phase-contrast light-microscopy photographs were taken at the same magnification (320×) for each specimen at each time point. The cell-survival index was computed for each specimen as the sum of the cells counted on the eight photographs. The number of elongated, flat and round fibroblasts present in each photograph was also revealed and their relative percentages were calculated at each experimental time for all samples. Proliferation was evaluated by the method of Kjellstrand et al. [9] using semiquantitative scoring (0, Isolated fibroblasts; 1, Isolated clusters; 2, Semiconfluence, i.e. large areas covered by clusters; 3, Confluence; i.e. complete coverage)

2.4. Scanning electron microscopy (SEM)

SEM (Philips SEM 515, Eindhoven, The Netherlands) was performed to examine the surfaces of the laser-welded and soldered joints and to further assess fibroblast morphology on day 16 after seeding. Specimens were fixed in 2.5% glutaraldehyde plus 4% paraformaldehyde dissolved in 0.1 
Na-cacodylate buffer, and post-fixed in osmium tetroxide dissolved in 0.1 
Na-cacodylate buffer. They were then dehydrated in ethanol and dried with hexamethyldisilazane (HDMS). Specimens were mounted on stubs with carbon biadhesive film and covered with a 20 nm-thick gold-palladium film and observed at 15 kV.

2.5. Statistical analysis

Because of abnormal distribution of the data and small data sets, non-parametric exact tests with Monte Carlo methods to compute probability were used. In particular, Kruskal-Wallis' Anova exact test was used to compare simultaneously laser-welding, soldering and control data on cell adhesion. Mann-Whitney's U test was used to compare data of welding and soldering with those of the control.

5. 3. Results

Significant differences in counts of survival fibroblasts were observed at all experimental times (Kruskal-Wallis' Anova P<0.001) (Table 1).

Table 1. Fibroblast adhesion index on soldering (S), laser-welding (L) and control (C) samples after 6 h, 24 h, 7 days and 16 days of culture

Kruskal-Wallis' Anova exact test with Monte Carlo Method to compute probability.

After 6 h in cultures, fewer cells, were noted on the laser-welded and soldered joints' surfaces with respect to the control (welding vs. control, Mann-Whitney's U test P<0.05; soldering vs. control , Mann-Whitney's U test P<0.05). The same phenomenon was noted after 24 h (welding vs. control, Mann-Whitney's U test P<0.05; soldering vs. control, Mann-Whitney's U test P<0.05).

In contrast, at days 7 and 16, only the soldered joints surfaces showed significantly fewer fibroblasts than the control (P<0.05 and P<0.001, respectively) whereas the laser-welded ones showed no differences from the control.

After 6 h of culture, fewer elongated cells were observed on both the laser-welded (P<0.05) and soldered surfaces (P<0.05) than onto the controls. No significant differences were observed as far as the number of round and flat cells were concerned.

This phenomenon was also observed at 24 h on laser-welded joint surfaces counted (P<0.05) but after that time, no differences were further noted for any of the morphotypes with respect to the control.

In contrast, fewer elongated cells (indicating less adhesion) were always observed either at 24 h, 7 day, and 16 day intervals on soldered substrates with respect to the control (24 h: P<0.001; 7 days: P<0.001; 16 days: P<0.001). Rounded cells were never observed on the control at 24 h, 7 day, and 16 day intervals whereas they were always present on soldered surfaces with an increasing trend over time. Relative prevalences of the morphology of fibroblasts adhering onto the substrates are reported in Fig. 1.

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Fig. 1. Fibroblast adhesion on substrate near soldering (S) and laser-welded (L) samples and in controls (C) after 6 h, 24 h, 7 days and 16 days of culture.

Fig. 2 shows representative contrast-phase light microscopy (320×) images in each setting at the various time points. The fibroblasts on both the laser-welded and control substrates showed similar patterns: at 6 h, frequent clusters of elongated cells were interspersed with residual round cells; at 24 h, they almost all showed an elongated shape and were semiconfluent; at 7 and 16 days, fibroblasts were all elongated and confluent. The soldered substrates showed a different pattern: at 6 h, isolated elongated cells were outnumbered by round cells; at 24 h, the elongated cells were isolated and associated with frequent round cells; at 7 and 16 days, the elongated cells remained isolated in a context of an increasing percentage of round cells. As can be seen from Table 2 proliferation scores were higher on both laser-welded samples and in control cultures with respect to the soldered samples.

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Fig. 2. Representative contrast-phase light microscopy (320×) images of human gingival fibroblasts on substrate near soldered (S) and laser-welded (L) samples and in controls (C) after (a) 6 h, (b) 24 h, (c) 7 days and (d) 16 days of culture.

Table 2. Fibroblast proliferation scores on substrate near soldering (S) and laser-welding (L) samples and in controls (C) after 6 h, 24 h, 7 days and 16 days of culture

Score 0, Isolated; Score 1, Clusters; Score 2, Semiconfluence; Score 3, Confluence.

Fig. 3 shows SEM imaging of the different morphologies of laser-welded and soldered joints after 16 days of culture. On the laser-welded joints, elongated fibroblasts were observable at confluence. By contrast, on the soldered ones, only fibroblast debris was observable, as a clear consequence of cytotoxicity.

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Fig. 3. (a) Fibroblast adhesion on the laser-welded joint in contrast to (b) cell debris on the soldered joint (SEM bar 100 
m).

6. 4. Discussion

The biocompatibility of dental components is a critical issue because of the long-term intimate contact with oral tissues. Studies have evaluated the adhesion, proliferation and metabolism of cells (Balb/c, 3T3, L929, WI38, human fibroblasts, osteoblasts) cultivated either in direct contact with orthodontic materials [4, 10, 11, 12 and 13], or in media left in contact with dental components [14, 15 and 16]. Some studies have also assessed the types and quantities of ions released from single component and casting alloys in culture media over time [2, 4, 12, 17 and 18].

Thus, studies are also required on the biocompatibility of joints between components, where alterations in the physical and chemical properties of the orthodontic materials could lead to adverse cell reactions.

In this work, using the "in vitro" technique, we characterised some phenotypic expressions of fibroblasts obtained from the human oral mucosa cultived on two different types of orthodontic joints.

We found that the reaction of the fibroblasts to the laser-welded samples was similar to that of the control substrate: the cells rapidly acquired and subsequently retained their typical elongated shape, showing no sign of cell suffering at either light microscopy or SEM. By contrast, on the substrate of the soldered samples the fibroblasts showed no sign of adaptation at any time during the study; at 16 days, SEM revealed cell debris on the joint, a clear indication of cytotoxicity which could depend on several causes. The effects of the direct contact with the joints' surface depend on its shape and consistency (which appear to be major factors in the release of ions [19]), as well as its chemical properties after soldering. Different elements have different liabilities (or tendencies to be released); elements such as Zn, Cu and Ni, which are all present in soldering supply material, are more labile than other metals used in higher concentration in dental alloys [20]. Another potential cause of cytotoxicity is the release of metallic ions as a consequence of corrosion, to which copper-based brazing alloys are known to be particularly subject [2].

The in vitro nature of our main findings limits the possibility of drawing clinical conclusions. Further studies are needed to assess the clinical implications of our in vitro findings regarding the superior fibroblast biocompatibility of laser-welded joints and the potential cytotoxic effects of the soldered ones.

7. 5. Conclusion

We analysed in vitro the adhesion and proliferation of human gingival fibroblasts (important components of the gum that readily reveal signs of inflammation) placed in direct contact with conventionally soldered and laser-welded joints for up to 16 days.

To our knowledge, this is the first cell-culture study to address the surface biocompatibility of the joints of dental components. The results of our in vitro study on the reactions of human fibroblasts placed in direct contact with the joints highlights the superior fibroblast biocompatibility of laser-welding over soldering.

8. Acknowledgements

We are grateful to Robin M.T. Cooke for helping prepare the manuscript and to Dentaurum Italia for financial support.

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