10.59987/ads/2013.3-4.244-249
Aim. To evaluate the growth of Human Gingival Fibroblasts (HGFs) cultured onto sample discs of CAD/CAM zirconia and veneering ceramic for zirconia by means of Scanning Electron Microscope (SEM) analysis at different experimental times.
Methods. A total of 26 experimental discs, divided into 2 groups, were used: Group A) CAD/CAM zirconia (3Y-TZP) discs (n=13); Group B) veneering ceramic for zirconia discs (n=13). HGFs were obtained from human gingival biopsies, isolated and placed in culture plates. Subsequently, cells were seeded on experimental discs at 7,5x103/cm2 concentration and cultured for a total of 7 days. Discs were processed for SEM observation at 3h, 24h, 72h and 7 days.
Results. In Group A, after 3h, HGFs were adherent to the surface and showed a flattened profile. The disc surface covered by HGFs resulted to be wider in Group A than in Group B samples. At SEM observation, after 24h and 72h, differences in cell attachment were slightly noticeable between the groups, with an evident flattening of HGFs on both surfaces. All differences between Group A and group B became less significant after 7 days of culture in vitro.
Conclusions. SEM analysis of HGFs showed differences in terms of cell adhesion and proliferation, especially in the early hours of culture. Results showed a better adhesion and cell growth in Group A than in Group B, especially up to 72h in vitro. Differences decreased after 7 days, probably because of the rougher surface of CAD/CAM zirconia, promoting better cell adhesion, compared to the smoother surface of veneering ceramic.
application/pdf
1971-1441
131
3-4
en
S. Tetè
https://creativecommons.org/licenses/by-nc-nd/4.0
Annali di stomatologia
ceramic cell attachment
SEM evaluation of human gingival fibroblasts growth onto CAD/CAM zirconia and veneering ceramic for zirconia
Text.Serial.Journal
https://www.annalidistomatologia.eu/ads
4
Original Article
2022-08-16
2022-08-16
2013-12-01
2023-12-04
244-249
Ann Stomatol (Roma)
S. Tetè
2013
10.59987/ads/2013.3-4.244-249
244
1971-1441
3-4
ceramic cell attachment
en
249
SEM evaluation of human gingival fibroblasts growth onto CAD/CAM zirconia and veneering ceramic for zirconia
https://www.annalidistomatologia.eu/ads/article/download/131/114
4
4_Tetè_Riv Annali 3-4 2013 13/02/14 10:45 Pagina 244




                                                                                                                       Original article



                   SEM evaluation of human gingival fibroblasts
                   growth onto CAD/CAM zirconia and veneering
                   ceramic for zirconia




                                                                                                                                          li
                                                                                    na
                   Vincenzo Zizzari, DDS1                                      to the surface and showed a flattened profile. The
                   Bruna Borelli, DDS2                                         disc surface covered by HGFs resulted to be wider




                                                                                  io
                   Marianna De Colli, PhD1                                     in Group A than in Group B samples. At SEM obser-
                   Margherita Tumedei, DDS3                                    vation, after 24h and 72h, differences in cell attach-
                   Donato Di Iorio, DDS3                                       ment were slightly noticeable between the groups,
                   Susi Zara, PhD1                                             with an evident flattening of HGFs on both surfaces.




                                                                               az
                   Roberto Sorrentino, DDS, MSc, PhD2                          All differences between Group A and group B be-
                   Amelia Cataldi, MD1                                         came less significant after 7 days of culture in vitro.
                   Enrico Felice Gherlone4                                     Conclusions. SEM analysis of HGFs showed differ-
                   Fernando Zarone, MD, DDS2                                   ences in terms of cell adhesion and proliferation,




                                                                            rn
                   Stefano Tetè, DDS3                                          especially in the early hours of culture. Results
                                                                               showed a better adhesion and cell growth in Group
                                                                               A than in Group B, especially up to 72h in vitro. Dif-
                   1 Department of Pharmacy, “G. D’Annunzio” University        ferences decreased after 7 days, probably because




                                                                         te
                     of Chieti-Pescara, Italy                                  of the rougher surface of CAD/CAM zirconia, pro-
                   2 Department of Neurosciences and Reproductive and          moting better cell adhesion, compared to the
                     Odontostomatological Sciences, “Federico II” Univer-      smoother surface of veneering ceramic.
                     sity of Naples, Italy
                   3 Department of Medical, Oral, and Biotechnological
                                                                      In       Key words: CAD/CAM zirconia, veneering ceramic,
                     Sciences, University “G. D’Annunzio” University of        human gingival fibroblasts, scanning electron mi-
                     Chieti-Pescara, Italy                                     croscope, zirconia cell attachment, ceramic cell at-
                   4 Department of Dentistry, “Vita Salute S. Raffaele” Uni-   tachment.
                                                        ni
                     versity, Milan, Italy

                                                                               Introduction
                   Corresponding author:
                                           o

                   Stefano Tetè                                                In the last decades, the introduction of metal-free
                   Department of Medical, Oral,                                restorations has led to the development of innovative
                                       izi


                   and Biotechnological Sciences                               ceramic materials showing excellent optical properties
                   “G. D’Annunzio” University                                  and better mechanical characteristics compared to the
                   Via dei Vestini, 31                                         early dental ceramics; besides, the introduction of
                   66100 Chieti, Italy                                         Computer Aided Design/Computer Aided Manufactur-
                          Ed




                   Phone: +39 0871 3554095                                     ing (CAD/CAM) technologies has allowed high preci-
                   E-mail: tete@unich.it                                       sion and predictability of the restorative results. The
                                                                               most noticeable advantages of the metal-free materials
                                                                               are: translucency, esthetic natural appearance, chro-
                   Summary                                                     matic stability, low plaque retention and fluids absorp-
                                                                               tion, high hardness, wear resistance, low thermal con-
               C




                   Aim. To evaluate the growth of Human Gingival Fi-           ductivity and chemical inertness (1).
                   broblasts (HGFs) cultured onto sample discs of              Many in vitro and clinical studies reported optimum bio-
    CI




                   CAD/CAM zirconia and veneering ceramic for zirco-           compatibility for high strength polycristalline ceramics
                   nia by means of Scanning Electron Microscope                (e.g. alumina, zirconia), showing favorable biological
                   (SEM) analysis at different experimental times.             responses in soft tissues. In particular, the use of zirco-
                   Methods. A total of 26 experimental discs, divided in-      nia has become more and more widespread in the clini-
                   to 2 groups, were used: Group A) CAD/CAM zirconia           cal practice, for the fabrication of single crowns, fixed
                   (3Y-TZP) discs (n=13); Group B) veneering ceramic           dental prostheses and implant abutments (2-5).
©




                   for zirconia discs (n=13). HGFs were obtained from          Prolonged contact between these prostheses and oral
                   human gingival biopsies, isolated and placed in cul-        soft tissues makes the biocompatibility and the integra-
                   ture plates. Subsequently, cells were seeded on ex-         tion of these materials critical for long-term success (6,
                   perimental discs at 7,5x103/cm2 concentration and           7). Several all-ceramic materials and surface modifica-
                   cultured for a total of 7 days. Discs were processed        tion methods were proposed in order to improve bio-
                   for SEM observation at 3h, 24h, 72h and 7 days.             compatibility and soft tissue integration of fixed dental
                   Results. In Group A, after 3h, HGFs were adherent           restorations (1, 3).

                   244                                                                         Annali di Stomatologia 2013; IV (3-4): 244-249
4_Tetè_Riv Annali 3-4 2013 13/02/14 10:45 Pagina 245




                   SEM evaluation of human gingival fibroblasts growth onto CAD/CAM zirconia and veneering ceramic for zirconia


                   Some in vitro and in vivo studies on animal models              severe illness, unstable diabetes, drug abuse, history of
                   showed that the interaction between gingival fibroblast         head and neck irradiation, chemotherapy. Moreover,
                   cells and zirconia surface depends on a number of vari-         each subject was pretreated for 1 week with professional
                   ables related to the surface microtopography, the               dental hygiene and antibiotic therapy was administered
                   chemical composition and the cell phenotype charac-             pre-operatively (amoxicillin/clavulanic acid, 2 g 1 hour




                                                                                                                                          li
                   teristics (8). It was shown that the surface roughness          before extraction). The tissue fragments were immedi-
                   might alter cellular activity in vitro (9). This could be ac-   ately placed in Dulbecco’s modified Eagle’s medium




                                                                                       na
                   counted not only for the chemical and biological proper-        (DMEM) for at least 1 h, rinsed 3 times in phosphate-
                   ties but also for the structure of the surface, as it is        buffered saline solution (PBS), minced into small tissue
                   known that fibroblasts show greater affinity for smooth         pieces and cultured in DMEM containing 10% foetal
                   or finely grooved surfaces than for rough ones (10, 11).        bovine serum (FBS), 10% penicillin and streptomycin




                                                                                     io
                   The aim of the present investigation was to evaluate            and 1% fungizone. Cells were maintained at 37°C in a
                   the growth and cell attachment of Human Gingival Fi-            humidified atmosphere of 5% (v/v) CO2. Cultured HGFs
                   broblasts (HGFs) onto samples of CAD/CAM zirconia               with DMEM containing 10% FBS, 1% penicillin and
                   and veneering ceramic for zirconia at different experi-         streptomycin and following 4-8 passages were used.




                                                                                  az
                   mental times by means of Scanning Electron Micro-               Subsequently, each experimental disc was placed in a
                   scope (SEM) morphologic and qualitative analysis.               12-well plate and HGFs were seeded in each well at
                                                                                   7,5×103/cm2 concentration and cultured for a total of 7
                                                                                   days. HGFs at same concentration were also seeded in




                                                                               rn
                   Methods                                                         an empty well as control.
                                                                                   A qualitative analysis was performed under SEM for all
                   A total of 26 experimental discs were prepared for this         the discs at different experimental times. After 3 h, 24




                                                                            te
                   in vitro study. Thirteen discs of CAD/CAM yttria-stabi-         h, 72 h and 7 days in vitro, test discs were fixed in glu-
                   lized tetragonal zirconia (3Y-TZP) (IPS e.max Zir CAD,          taraldehyde 2% in 0.1 M phosphate buffer pH 7.2,
                   Ivoclar Vivadent AG, Liechtenstein, ISO standard                rinsed with phosphate buffer 0,15 M, dehydrated in an
                   13356. 1997) were fabricated in a milling center without        increasing ethanol series and finally dried in hexam-
                   receiving any surface treatment (Group A), while other
                   13 discs of veneering ceramic for zirconia were ob-
                                                                         In        ethyldisilazane. The samples were then metallized with
                                                                                   gold in a sputtering device and observed by SEM at
                   tained by die-casting in a laboratory and then polished         100x, 800x and 1600x magnification. Cell morphology
                   and glazed (Group B). All the samples had a mean sur-           was assessed on micrographs randomly taken indiffer-
                   face of 2,8 cm2. The discs were cleaned and disinfect-          ent areas of each experimental discs. The trial was
                                                             ni
                   ed by ultrasonic treatment in Alconox®-water solution           conducted in triplicate for each experimental point.
                   for 5 min; then, they were rinsed with sterile purified
                   water (cell-culture grade) and ultrasonically treated
                   again for 5 min in isopropyl alcohol. The samples for           Results
                                              o

                   HGFs culture were then transferred aseptically to sterile
                   12-well cell-culture trays and submerged in isopropyl al-       SEM observation of CAD/CAM zirconia discs showed a
                                          izi


                   cohol for 20 min, rinsed twice with sterile purified water      surface with regular roughness related to the milling
                   and dried for a minimum of 8 h at 60° C under aseptic           procedure of manufacturing, while discs of veneering
                   conditions. These procedures of disinfection were con-          ceramic showed a smooth surface with only little de-
                   gruent with previously published techniques for testing         fects, probably due to the glazing treatment (Figs. 1-3).
                           Ed




                   ceramic materials (12).                                         SEM analysis of HGFs on the different experimental
                   One disc per group was randomly selected and ana-               surfaces showed differences in terms of cell adhesion
                   lyzed by Scanning Electron Microscope (SEM Zeiss                and growth.
                   EVO-50, Cambridge, UK) for surface morphology ob-               Group A revealed HGFs almost completely adherent to
                   servation.                                                      the surface with a flattened profile and rather elongated
                   HGFs were obtained from fragments of healthy margin-            morphology, already after 3 h in vitro (Fig. 4). More-
               C




                   al gingival tissue from the retromolar area taken during        over, at the same time, the area covered by HGFs re-
                   surgical extraction of impacted third molars in adult           sulted higher in Group A than in Group B. HGFs in
    CI




                   subjects (aged 18 to 60). Each patient gave written in-         Group B discs appeared round shaped, flattened on the
                   formed consent for participating in this study as donor         surface with round nuclei. After 24 h of culture, both
                   of HGFs in accordance with the Local Ethics Commit-             Group A and Group B samples showed HGFs with de-
                   tee, in compliance with Italian legislation and the code        finitive morphology, long cytoplasmic elongations, even
                   of Ethical Principles for Medical Research involving Hu-        if a reduced area was covered by HGFs in Group B
                   man Subjects of the World Medical Association (Decla-           discs (Fig. 5).
©




                   ration of Helsinki).                                            After 72 h in vitro culture, SEM observation showed an
                   Before gingival tissue withdrawal, each subject under-          evident flattening of HGFs on both surfaces and differ-
                   went complete medical anamnesis for systemic and oral           ences in surface coverage could be not noticed be-
                   infections or diseases. All the selected patients had           tween the groups at that experimental time (Fig. 6). Af-
                   healthy systemic conditions, including the absence of           ter 7 days of culture, both surfaces appeared entirely
                   any diseases that would contraindicate oral surgery. The        covered by HGFs and no differences between the
                   exclusion criteria were: uncontrolled periodontal disease,      groups could be evidenced (Fig. 7).

                   Annali di Stomatologia 2013; IV (3-4): 244-249                                                                        245
4_Tetè_Riv Annali 3-4 2013 13/02/14 10:45 Pagina 246




                   V. Zizzari et al.



                      A                                                           B




                                                                                                                                             li
                                                                                    na
                                                                                  io
                                                                               az
                      C                                                           D




                                                                            rn
                                                                         te
                                                                      In
                                                         ni

                   Figure 1. Scanning electron micrographs analysis of CAD/CAM zirconia disc sample: A) 100x magnification; B) 800x magni-
                   fication; C) 1.60Kx magnification; D) Graphic representation of the surface roughness.
                                           o

                     A                                                B
                                       izi
                           Ed
               C




                     C                                                D
    CI
©




                   Figure 2. Scanning electron micrographs analysis of veneering ceramic for zirconia disc sample: A) 100x magnification; B)
                   800x magnification; C) 1.60Kx magnification; D) Graphic representation of the surface roughness.

                   246                                                                            Annali di Stomatologia 2013; IV (3-4): 244-249
4_Tetè_Riv Annali 3-4 2013 13/02/14 10:45 Pagina 247




                   SEM evaluation of human gingival fibroblasts growth onto CAD/CAM zirconia and veneering ceramic for zirconia




                                                                                                                                         li
                                                                                    na
                                                                                  io
                   Figure 3. Graphic 3D reconstruction of the surface morphology: A) CAD/CAM zirconia disc sample; B) veneering ceramic for
                   zirconia disc sample.




                                                                               az
                                                                            rn
                                                                         te
                                                                      In
                                              o              ni

                   Figure 4. Scanning electron micrographs of HGFs cultured for 3 h on the different experimental discs at 100x, 800x, and
                                          izi


                   1600x magnification: A) CAD/CAM zirconia disc sample; B) veneering ceramic for zirconia disc sample.
                           Ed
               C
    CI
©




                   Figure 5. Scanning electron micrographs of HGFs cultured for 24 h on the different experimental discs at 100x, 800x, and
                   1600x magnification: A) CAD/CAM zirconia disc sample; B) veneering ceramic for zirconia disc sample.

                   Annali di Stomatologia 2013; IV (3-4): 244-249                                                                      247
4_Tetè_Riv Annali 3-4 2013 13/02/14 10:45 Pagina 248




                   V. Zizzari et al.




                                                                                                                                              li
                                                                                     na
                                                                                   io
                                                                                az
                                                                             rn
                   Figure 6. Scanning electron micrographs of HGFs cultured for 72 h on the different experimental discs at 100x, 800x, and




                                                                          te
                   1600x magnification: A) CAD/CAM zirconia disc sample; B) veneering ceramic for zirconia disc sample.

                                                                       In
                                           o             ni
                                       izi
                           Ed
               C




                   Figure 7. Scanning electron micrographs of HGFs cultured for 7 days on the different experimental discs at 100x, 800x, and
                   1600x magnification: A) CAD/CAM zirconia disc sample; B) veneering ceramic for zirconia disc sample.
    CI




                   Discussion and conclusion                                      Zirconia has been used in the last years as an excellent
                                                                                  biomaterial for orthopedic and oral applications. The
                   Despite the complexity of cells and dental ceramic in-         addition of yttria as an allotropic transformation catha-
                   teraction in vivo, which is governed by a number of            lytic stabilizer was also investigated, resulting in a stabi-
©




                   chemical and physical processes, in vitro studies may          lized zirconia ceramic (3Y-TZP), exhibiting well demon-
                   be very useful for a comprehensive material selection          strated medium/long term mechanical performances
                   to achieve optimal soft tissue integration. Among the          when used for the fabrication of crown/bridge frame-
                   variables related to the structure and composition of          works (4, 5, 13-15). Due to a fairly high incidence of
                   biomaterials, one of the main topics is surface topogra-       mechanical complications, like the veneer ceramic chip-
                   phy, as it reflects in vitro cell behavior in term of cell     ping (16), in the last years the dental research has
                   morphology, proliferation and adhesion.                        been increasingly focused on the possibility of using

                   248                                                                             Annali di Stomatologia 2013; IV (3-4): 244-249
4_Tetè_Riv Annali 3-4 2013 13/02/14 10:45 Pagina 249




                   SEM evaluation of human gingival fibroblasts growth onto CAD/CAM zirconia and veneering ceramic for zirconia


                   zirconia as a monolithic material, for anatomically-                     J Dent 2007; 35:819-826.
                   shaped restorations, without the less fracture-resistant              3. Denry I, Kelly JR. State of the art of zirconia for dental ap-
                   feldspathic veneering ceramic.                                           plications. Dent Mater 2008; 24:299-307.
                                                                                         4. Sorrentino R, Galasso L, Tetè S, De Simone G, Zarone F.
                   The CAD/CAM zirconia discs appeared to be rougher
                                                                                            Clinical evaluation of 209 all-ceramic single crowns cemented
                   than the veneering ceramic ones. The evidenced fewer




                                                                                                                                                        li
                                                                                            on natrural and implant-supported abutments with different
                   and slower HGFs attachment to veneering ceramic,                         luting agents: a 6-year retrospective study. Clin Implant Dent
                   compared to that to zirconia surface, could be ex-




                                                                                            na
                                                                                            Relat Res 2012; 14:184-197.
                   plained considering that a rough surface could favor fi-              5. Sorrentino R, De Simone G, Tetè S, Russo S, Zarone F. Five-
                   broblasts adhesion and induce more rapidly the typical                   year prospective clinical study of posterior three-unit zirco-
                   flattened phenotype. In fact, HGFs showed a better ad-                   nia-based fixed dental prostheses. Clin Oral Investig 2012;
                   hesion and growth on CAD/CAM zirconia rather than                        16:977-985.




                                                                                          io
                   on ceramic veneer discs surface up to 24 h of in vitro                6. Messer RL, Lockwood PE, Wataha JC, Lewis JB, Norris S,
                                                                                            Bouillaguet S. In vitro cytotoxicity of traditional versus con-
                   culture. These findings suggested that a different sur-
                                                                                            temporary dental ceramics. J Prosthet Dent 2003; 90:452-
                   face roughness can affect HGFs adhesion and growth
                                                                                            458.




                                                                                       az
                   onto the different samples. After 24 h of culture, differ-            7. Li J, Liu Y, Hermansson L, Söremark R. Evaluation of bio-
                   ences in cell attachment between Group A and Group                       compatibility of various ceramic powders with human fi-
                   B discs became less evident, thus suggesting that, af-                   broblasts in vitro. Clin Mater 1993; 12:197-201.
                   ter an initial phase of adaptation, HGFs proliferated sig-            8. Piconi C, Maccauro G. Zirconia as a ceramic biomaterial. Bio-




                                                                                    rn
                   nificantly faster on the smooth veneering ceramic re-                    materials 1999; 20:1-225.
                   spect to the rougher surface of CAD/CAM zirconia, as                  9. Brunette DM. The effect of implant surface topography on
                   reported by Yamano et al. (17).                                          the behavior of cells. Int J Oral Maxillofac Implants 1988;
                                                                                            3:231-246.
                   For successful tissue integration to prosthetic ceramic
                                                                                        10. Chehroudi B, Gould TR, Brunette DM. A light and electron




                                                                                 te
                   materials, cells have to uniformly colonize the surface.
                                                                                            microscopic study of the effects of surface topography on the
                   Since after 24h of culture both CAD/CAM zirconia and                     behavior of cells attached to titanium-coated percutaneous
                   veneering ceramic materials favored an effective HGFs                    implants. J Biomed Mater Res 1991; 25:387-405.
                   proliferation process, the results of the present in vitro           11. Könönen M, Hormia M, Kivilahti J, Hautaniemi J, Thesleff I.
                   investigation allowed to hypothesize that both the test-
                                                                              In            Effect of surface processing on the attachment, orientation,
                                                                                            and proliferation of human gingival fibroblasts on titanium.
                   ed surfaces could be suitable to support in vivo soft tis-
                   sue integration. Further in vitro and clinical studies will              J Biomed Mater Res 1992; 26:1325-1341.
                   be needed to support the present findings.                           12. Messer RL, Lockwood PE, Wataha JC, Lewis JB, Norris S,
                                                                                            Bouillaguet S. In vitro cytotoxicity of traditional versus con-
                                                                                            temporary dental ceramics. J Prosthet Dent 2003; 90:452-
                                                              ni

                                                                                            458.
                   Acknowledgements                                                     13. Luthardt RG, Holzhüter M, Sandkuhl O, Herold V, Schnapp
                                                                                            JD, Kuhlisch E, et al. Reliability and properties of ground Y-
                                              o

                   The authors wish to thank FIRB-Accordi di Program-                       TZP-zirconia ceramics. J Dent Res 2002; 81:487-491.
                   ma 2010, “Processi degenerativi dei tessuti mineraliz-               14. Ambré MJ, Aschan F, von Steyern PV. Fracture strength of
                   zati del cavo orale, impiego di biomateriali e controllo                 yttria-stabilized zirconium-dioxide (Y-TZP) fixed dental pros-
                                          izi



                   delle interazioni con i microrganismi dell’ambiente”,                    theses (FDPs) with different abutment core thicknesses and
                   for the fellowship attributed to Dr. V.L. Zizzari and Dr.                connector dimensions. J Prosthodont 2013 Jan 4. doi:
                                                                                            10.1111/jopr.12003. [Epub ahead of print].
                   M. De Colli; dental technician Mr. Attilio Sommella and
                                                                                        15. Takaba M, Tanaka S, Ishiura Y, Baba K. Implant-support-
                           Ed




                   IVOCLAR Vivadent for providing the sample disks.                         ed fixed dental prostheses with CAD/CAM-fabricated porce-
                                                                                            lain crown and zirconia-based framework. J Prosthodont 2013
                                                                                            Jan 4. doi: 10.1111/jopr.12001.
                   References                                                           16. Kimmich M, Stappert CF. Intraoral treatment of veneering
                                                                                            porcelain chipping of fixed dental restorations: a review and
                    1. Zarone F, Russo S, Sorrentino R. From porcelain-fused-to-            clinical application. J Am Dent Assoc 2013; 144:31-44.
                                                                                        17. Yamano S, Ma AK, Shanti RM, Kim SW, Wada K, Sukotjo
               C




                       metal to zirconia: clinical and experimental considerations.
                       Dent Mater 2011; 27:83-96.                                           C. The influence of different implant materials on human gin-
                    2. Manicone PF, Rossi Iommetti P, Raffaelli L. An overview of           gival fibroblast morphology, proliferation, and gene expres-
    CI




                       zirconia ceramics: basic properties and clinical applications.       sion. Int J Oral Maxillofac Implants 2011; 26:1247-1255.
©




                   Annali di Stomatologia 2013; IV (3-4): 244-249                                                                                     249
