Evaluation of cell morphology and adhesion capacity of human gingival fibroblasts on titanium discs with different roughened surfaces: an in vitro scanning electron microscope analysis and cell culture study

Background: Implantoplasty is an option in peri-implantitis treatment. What is known about the effects of implantoplasty on peri-implant soft tissue adhesion and cell behaviors is limited. This study aimed to evaluate the morphological features and adhesion capacity of human gingival fibroblast (HGF) cells onto sand-blasted, large-grit, acid-etched (SLA®) titanium (Ti) discs surfaces roughened with different implantoplasty protocols. Materials and methods: The study included a total of 48 Ti discs divided into four groups (n=12) per group: Group I: machined, smooth surface discs; Group II: SLA® surface discs; Group III: SLA® surface discs roughened with diamond bur sequence (40 and 15-μm grit); Group IV: SLA® surface discs roughened with diamond bur sequence (125 and 40-μm grit). Following polishing procedure, the surface roughness value of discs were assessed by a profilometer and scanning electron microscope (SEM). HGFs were cultured on Ti discs and cell adhesion was examined after the 24th, 48th, and 72nd hours. Statistical significance was set at the p≤0.05 level. Results: SEM analyses of the discs revealed that fibroblasts exhibited well-dispersion and a firm attachment in all groups. The cells in group I and II had thin and long radial extensions from the areas where the nucleus was located to the periphery; however attached cells in group III and IV showed more spindle-shaped morphology. The surface roughness parameters of the test groups were lower than those of the SLA®. The SLA® group showed the highest HGF adhesion (group II) (p≤0.05). HGF adhesion in group IV was greater compared to group III, but less than group I. Conclusions: This study showed that the characteristics of the burs applied in the implantoplasty protocol are determinant for the surface roughness and fibroblast adhesion occurs on surfaces with decrased roughness following implantoplasty. Consequently, it should be kept in mind that the surface properties of the implant may affect the adherent cell morphology and adhesion

Free titanium particles act synergistically with P. gingivalis lipopolysaccharide - An in vitro experimental study of periimplantitis

Τις τελευταίες δύο δεκαετίες, η σημαντική αύξηση των ασθενών που φέρουν εμφυτεύματα φέρνει τον κλινικό αντιμέτωπο με την πρόληψη και θεραπεία της περιεμφυτευματικής παθολογίας, καθώς και με τη μακροπρόθεσμη διατήρηση του θεραπευτικού αποτελέσματος. Τα τελευταία χρόνια, το ενδιαφέρον της ερευνητικής κοινότητας στρέφεται προς τα ελεύθερα σωματίδια τιτανίου, ως προϊόντα διαβρωτικών ή αποτριπτικών διεργασιών στην επιφάνεια του τιτανίου, είτε κατά τη χειρουργική θεραπεία της περιεμφυτευματίτιδας ή από διάφορες παρεμβάσεις κατά την εφαρμογή του προληπτικού προγράμματος. Το ερώτημα που τίθεται είναι, κατά πόσον αυτά τα σωματίδια είναι ικανά να προκαλέσουν ή να εντείνουν μια ήδη εδραιωμένη φλεγμονώδη αντίδραση των ιστών, γεγονός που σίγουρα δεν βοηθάει την ίαση μίας υπάρχουσας νοσολογικής οντότητας. Σκοπός Σκοπός της παρούσας διπλωματικής εργασίας είναι η διερεύνηση της επίδρασης των σωματιδίων στον ίδιο ή σε μεγαλύτερο βαθμό από το λιποπολυσακχαρίτη από P. Gingivalis, στη γονιδιακή έκφραση των προφλεγμονωδών κυτοκινών, ιντερλευκίνης-6 και ιντερλευκίνης-8, καθώς και του κολλαγόνου τύπου Ι σε ανθρώπινης προέλευσης ουλικές ινοβλάστες. Η πιθανότητα ύπαρξης μίας συνεργικής δράσης μεταξύ λιποπολυσακχαρίτη και σωματιδίων τιτανίου επίσης διερευνάται. Μέθοδοι και Υλικά Ανθρώπινης προέλευσης ουλικές ινοβλάστες καλλιεργήθηκαν σε αδροποιημένες και αμμοβολημένες (SLA) επιφάνειες τιτανίου, καθώς και σε επιφάνειες πολυστυρενίου για συνολικά 7 ημέρες. Ένα εικοσιτετράωρο μετά την καλλιέργεια των κυττάρων στις επιφάνειες, πραγματοποιήθηκε η έγχυση λιποπολυσακχαρίτη, σωματιδίων τιτανίου ή και των δύο μαζί στις πειραματικές ομάδες. Μετά από 24, 48 και 72 ώρες, διενεργήθηκε δοκιμασία ΜΤΤ για τον έλεγχο του κυτταρικού πολλαπλασιασμού. Επιπροσθέτως, για τις ίδιες χρονικές στιγμές, πραγματοποιήθηκε μικροσκοπία φθορισμού με χρώση FDA/PI για την αξιολόγηση της ζωτικότητας και απόπτωσης των κυττάρων. Στις 5 και 7 ημέρες, ολοκληρώθηκε η συνολική απομόνωση του RNA από όλες τις ομάδες για τον έλεγχο της γονιδιακής έκφρασης των ιντερλευκινών και κολλαγόνου, αλλά και η προετοιμασία των δίσκων για ηλεκτρονική μικροσκοπία σάρωσης. Αποτελέσματα Όσον αφορά στον πολλαπλασιασμό των κυττάρων, παρατηρήθηκε στατιστικά σημαντική διαφορά μεταξύ των δύο τύπων επιφανειών, με την αδρή επιφάνεια τιτανίου να ευνοεί λιγότερο τον πολλαπλασιασμό των ινοβλαστών. Παρόλα αυτά, ανεξάρτητα από το είδος της έγχυσης, όλες οι ομάδες παρουσίασαν στατιστικά σημαντική αύξηση του πολλαπλασιασμού μεταξύ των υπό εξέταση χρονικών στιγμών. Ως προς τη γονιδιακή έκφραση των ιντερλευκινών, ο συνδυασμός λιποπολυσακχαρίτη και σωματιδίων τιτανίου αυξάνει σημαντικά τα επίπεδα ιντερλευκίνης-8, σε σχέση με τη μονοθεραπεία με σωματίδια ή με ενδοτοξίνη. Επιπροσθέτως, στις επιφάνειες τιτανίου, η συνέργεια των δύο επέφερε σημαντική αύξηση τόσο της ιντερλευκίνης-6 όσο και του κολλαγόνου. Η μικροσκοπία σάρωσης αποκάλυψε την απώλεια συνδέσεων μεταξύ των κυττάρων, την επιμηκυμένη μορφολογία τους, καθώς και δυσκολία προσαρμογής τους στην επιφάνεια τιτανίου. Συμπεράσματα Οι αδρές επιφάνειες τιτανίου επιδρούν σημαντικά στον πολλαπλασιασμό και τη μορφολογία των ανθρωπίνων ουλικών ινοβλαστών, που υπό συνθήκες υγείας, δεν βρίσκονται σε επαφή με το αδρό τμήμα του εμφυτεύματος, παρά μόνο αν προκύψει οστική απορρόφηση. Ακόμη, ο συνδυασμός των σωματιδίων τιτανίου και του βακτηριακού λιποπολυσακχαρίτη επιφέρει στατιστικά σημαντική άυξηση των προφλεγμονωδών κυτοκινών, IL-6 και IL-8, καθώς και του κολλαγόνου τύπου Ι. Λαμβάνοντας υπόψιν τον ανοσορυθμιστικό ρόλο των ινοβλαστών στο συνδετικό ιστό του περιεμφυτευματικού βλεννογόνου, φαίνεται ότι τα ελεύθερα σωματίδια τιτανίου προκαλούν παρόμοια κυτταρική αντίδραση με μία μικροβιακή ενδοτοξίνη, ενώ συνεργικά εντείνουν αυτή την αντίδραση. Τέλος, καθώς τα ίδια κύτταρα είναι υπεύθυνα για τη σύνθεση κολλαγόνου και την παραγωγή της εξωκυττάριας θεμέλιας ουσίας, η αύξηση της έκφρασης του κολλαγόνου δυνητικά υποδηλώνει την αυξημένη δραστηριότητα των κυττάρων προς παραγωγή αντιδραστικού ιστού.The substantial increase of patients bearing implants, the past two decades, brings the clinician before the prevention and treatment of peri-implant pathology, as well as the long-term maintenance of the therapeutic outcome. The past years, the research interest has been turned towards free titanium particles, that are the product of corrosive events or the mechanical debridement of the implant surface, during the surgical therapy of periimplantitis or during the maintenance care program with several types of instruments. The question is whether these particles are capable of inducing or worsen an already established inflammatory reaction that will certainly not improve the condition, despite the good will of the clinician. Aim The aim of the present study is to investigate the effect of titanium particles, on the same or maybe greater degree than P. gingivalis lipopolysaccharide, on the gene expression of two major pro-inflammatory cytokines, interleukin-6 and interleukin-8, as well as gene expression of collagen type I of human gingival fibroblasts. The possibility of a synergistical activity between titanium particles and LPS is also examined. Materials and Methods Human gingival fibroblasts were cultured on sand blasted and acid-etched (SLA) titanium and TCP surfaces for 7 days. 24 hours after the culture of cells on both surfaces, the cells were treated with either LPS, particles or LPS and particles combined. At 24, 48 and 72 hours after treatment, MTT assay was performed to assess cell proliferation. FDA/PI staining has also been performed for the same time periods, in order to evaluate cell viability/apoptosis. At 5 and 7 days after the treatment, cell RNA was isolated and PCR was performed to assess gene expressions of IL-6, IL-8 and COL1A1. SEM microscopy was also performed five and seven days after the treatment for all groups on SLA surfaces. Results hGFs have shown a statistically significant difference in proliferation rates on SLA surfaces, compared to TCP, in favor of the latter. However, all groups, independently of the treatment kind, had a significant increase of their population between the time periods of examination. As far as the expression of interleukin levels is concerned, the combination of LPS and particles significantly increases the levels of interleukin-8 expression, compared to LPS or particles alone. On SLA surfaces, treatment with LPS and particles, induced a significant increase of interleukin-6 and collagen. FDA/PI microscopy has shown increased cell apoptosis for all treatment groups, as well as differences on cell morphology. SEM microscopy has revealed stressed cells with zones of no cells and a disrupted adhesion on SLA surfaces. Conclusions Rough titanium surfaces significantly affect the proliferation and morphology of human gingival fibroblasts, which contact such surfaces after bone resorption and pocket formation induced by inflammation. Moreover, the combination of titanium particles and LPS increase, in a statistically significant manner, the expression of IL-6, IL-8 and collagen. Taking into account the immunoregulatory role of fibroblasts in the connective tissue of the peri-implant mucosa, it appears that particles arouse a similar cellular reaction as the bacterial endotoxin, while synergistically intensifying this reaction. Finally, since these cells are also responsible for collagen synthesis and extracellular matrix production, the increase in collagen expression, indicates an increased activity of cells towards the production of reactive tissue

Laser treatment and disease characteristics of peri-implantitis

Peri-implantitis is an inflammatory disease which affects the soft and hard tissues surrounding dental implants. Current theory is that peri-implantitis is a counterpart to periodontitis, which is the inflammatory disease that destroys the tooth’s supporting tissues. However, there are some key discrepancies and there is a need for more studies on the characteristics of peri-implantitis. If the disease is not stopped, there is a risk that the dental implant is lost. It has proven difficult to treat peri-implantitis with conventional nonsurgical or surgical treatments. This is also an area of peri-implantitis that needs further research. The overall aim was to evaluate laser treatment of peri-implantitis, as well as explore the patients’ experiences and disease characteristics. In study I, we tested the safety of using diode lasers on dental implants by evaluating two different wavelengths 445 nm and 970nm. The temperature increase in dental implants were tested in two different models, one being a PM. Rigorous amounts of cooling water and limiting the continuous irradiation time to a maximum of 15-20s seemed to be key factors, depending on the power setting. We also assessed potential surface alterations on titanium discs in SEM but did not see any. In study II, the patient’s experiences and sensations were explored qualitatively in semi-structured interviews. We confirmed previous findings of positive associations of dental implants and added some perspectives to them with some negative experiences. In this group of patients, already diagnosed with peri-implantitis, some reported feeling symptoms, which could mean that with education on early signs of inflammation, the patient can also monitor their peri-implant health. The experiences of laser treatment and mucosal flap surgery were that of slight discomfort and for both the localized anesthesia was described as the worst part. Those that received surgery mentioned the sutures as a main source of discomfort. This we confirmed quantitatively in study III, where the surgery patients rated their discomfort significantly higher during the first week of healing than those in the laser group. In study III, we could not establish equivalence in change of PPD and RBL between the laser treatment and mucosal flap surgery. Both treatments had similar numbers of unresponsive peri-implantitis lesions, but in the patients that improved in their PPD, the surgery had significantly higher pocket reduction. There was basically no difference in change of RBL, BOP, biomarkers, and bacteria between the treatments. In study IV, we found that the inflammatory profile and immune cell composition were similar between peri-implantitis and periodontitis. In homogenized soft tissue, peri-implantitis lesions had significantly higher levels of IL-1β, TNF-α, IL-4 IL-17A, IL-23, G-CSF, and BAFF, whereas periodontitis only had significantly higher levels of IL-1β, IL-4, and G-CSF, compared to non-disease controls. Although, peri-implantitis and periodontitis did not significantly differ, there was a tendency towards a stepwise increase in proportion of B cells, from lowest in controls to highest in peri-implantitis. The results in this thesis show the potential for 970 nm diode laser in treating peri-implantitis. With unique clinical data a suggested role for the treatment modality could be initial stages of peri-implantitis where extensive pocket elimination is not as needed. The findings also support the need for further studies in assessing the patients self-monitoring of peri-implant conditions as well as the potential difference in B cell proportion and activity between peri-implantitis and periodontitis

Host-bacterial interaction in peri-implantitis

Crielaard, W. [Promotor]Loos, B.G. [Promotor]Wismeyer, D. [Promotor]Laine, M.L. [Copromotor

Hybrid materials for meniscus replacement in the knee

The meniscus is cartilage that not only prevents the bones in knee joints to grind together but acts as a joint stabiliser. Many athletes and older people suffer from meniscus tears and degeneration. Meniscal tear treatments have been through meniscal suture or by partial meniscectomy (removal). These treatments may cause changes in loading or decreased contact area and increased contact stress. Consequently, the ultimate result is a total meniscectomy that potentially leads to osteoarthritis (OA). These current surgical strategies have lower success rates in younger patients. There are no successful artificial meniscus replacement devices for young patients, therefore, new materials for meniscus replacement are required. Here, the aim was to develop a novel biomimetic meniscus device made of a silica/polytetrahydrofuran (SiO2/polyTHF) inorganic/organic hybrid material. The device is biomimetic in terms of its structural design, mechanical properties, and integration with the host tissue. The device should delay onset of OA. The hybrid has unique properties in that is a bouncy material which has comparable mechanical properties to knee cartilage. Two pot hybrid synthesis was used to synthesise the SiO2/polyTHF hybrid and casting mould was developed based on the shrinkage factor of the hybrid. The hybrid synthesis modifications were conducted by controlling compositions and drying processes. Biological fixation of the hybrid meniscus was achieved by titanium anchors with gyroid porous architecture which can provide initial mechanical fixation and secondary biological fixation on the tibia. The architecture was designed using Solidworks and Rhinoceros software and printed by the Additive Manufacturing technique of selective laser melting (SLM). Mechanical testing of the device included compression, cyclic loading, shear strength and long-term 90 days in-vitro mechanical testing, tribology against living bovine 2 cartilage, and cell studies. The results suggest that combination of hybrid and Ti gyroid has potential to be meniscus implant due to comparable mechanical properties, low friction coefficient, and non-cytotoxicity.Open Acces

Latest Trends in Surface Modification for Dental Implantology: Innovative Developments and Analytical Applications

An increase in the world population and its life expectancy, as well as the ongoing concern about our physical appearance, have elevated the relevance of dental implantology in recent decades. Engineering strategies to improve the survival rate of dental implants have been widely investigated, focusing on implant material composition, geometry (usually guided to reduce stiffness), and interface surrounding tissues. Although efforts to develop different implant surface modifications are being applied in commercial dental prostheses today, the inclusion of surface coatings has gained special interest, as they can be tailored to efficiently enhance osseointegration, as well as to reduce bacterial-related infection, minimizing peri-implantitis appearance and its associated risks. The use of biomaterials to replace teeth has highlighted the need for the development of reliable analytical methods to assess the therapeutic benefits of implants. This literature review considers the state-of-the-art strategies for surface modification or coating and analytical methodologies for increasing the survival rate for teeth restoration.Ministerio de Ciencia e Innovación PID2019-109371GB-I00Junta de Andalucía PAIDI 2020, P20_00671Universidad de Sevilla US-1380878, PPI505/2020, PPI532/202

Experimental implantoplasty outcomes correlate with fibroblast growth in vitro

Background Implantoplasty is an option in peri-implantitis treatment, but little is known about the effect on the soft tissue. The aim of the study was to characterize surface roughness following experimental implantoplasty and to examine its effect on human fibroblast growth and secretion of selected proteins. Methods Titanium grade IV coins were mechanically treated with six different rotating bur sequences; diamond burs or carbide burs alone, or followed by either Arkansas stone bur or silicone burs. Machined and rough-surface sandblasted, acid-etched (SLA) coins were used as control. The surface topography was characterized by scanning electron microscope and profilometer. Human gingival fibroblasts from two donors were cultured on the coins to quantify the effect on cell morphology, growth, and protein secretion by confocal microscopy and multiplex immunoassay. Results All surface roughness parameters were lower for the surfaces treated with experimental implantoplasty than for the SLA surface, and the sequence of carbide burs followed by silicone burs rendered the least rough surface of the test groups. The implantoplasty procedures changed the elemental composition of the titanium surface. High surface roughness showed a weak to moderate negative correlation to fibroblast growth, but induced a higher secretion of VEGF, IL-6 and MCP-3 to the cell medium compared to the least rough surfaces of the test groups. At day 30 fibronectin levels were higher in the SLA group. Conclusions The surface roughness following implantoplasty demonstrated a weak to moderate negative correlation with the growth of fibroblasts. The addition of Arkansas stone and silicon burs to the experimental implantoplasty bur protocol rendered an initial increase in fibroblast growth. Implantoplasty altered the elemental composition of the titanium surface, and had an effect on the fibroblast cytokine secretion and fibronectin levels