Marco Annunziata

The effects of titanium nitride-coating on the topographic and biological features of TPS implant surfaces

OBJECTIVES Titanium nitride (TiN) coating has been proposed as an adjunctive surface treatment aimed to increase the physico-mechanical and aesthetic properties of dental implants. In this study we investigated the surface characteristics of TiN-coated titanium plasma sprayed (TiN-TPS) and uncoated titanium plasma sprayed (TPS) surfaces and their biological features towards both primary human bone marrow mesenchymal stem cells (BM-MSC) and bacterial cultures. METHODS 15 mm×1 mm TPS and TiN-TPS disks (P.H.I. s.r.l., San Vittore Olona, Milano, Italy) were topographically analysed by confocal optical profilometry. Primary human BM-MSC were obtained from healthy donors, isolated and expanded. Cells were seeded on the titanium disks and cell adhesion, proliferation, protein synthesis and osteoblastic differentiation in terms of alkaline phosphatase activity, osteocalcin synthesis and extracellular mineralization, were evaluated. Furthermore, adhesion and proliferation of Streptococcus pyogenes and Streptococcus sanguinis on both surfaces were also analysed. RESULTS TiN-TPS disks showed a decreased roughness (about 50%, p < 0.05) and a decreased bacterial adhesion and proliferation compared to TPS ones. No difference (p > 0.05) in terms of BM-MSC adhesion, proliferation and osteoblastic differentiation between TPS and TiN-TPS surfaces was found. CONCLUSIONS TiN coating showed to modify the topographical characteristics of TPS titanium surfaces and to significantly reduce bacterial adhesion and proliferation, although maintaining their biological affinity towards bone cell precursors.

Effect of metronidazole and modulation of cytokine production on human periodontal ligament cells

Periodontitis is a multifactorial polymicrobial infection characterized by a destructive inflammatory process affecting tooth-supporting tissues and resulting in periodontal pocket formation, alveolar bone resorption and, eventually, tooth loss. The continuous challenge of host immune and resident cells by periodontopathogens and their virulence factors, such as lipopolysaccharide (LPS), results in enhanced and uncontrolled secretion of cytokines. The latter directly or indirectly participate in tissue destruction and bone resorption. Metronidazole (MTZ) is a widely used antimicrobial agent. The immunomodulatory effects of antibiotics might influence the degree of the local response to infection on the human periodontal ligament cell (HPLC). HPLCs play a role in the immune response of the oral cavity. In addition, HPLC can produce cytokines that increase the inflammatory response and that supply for normal communication. MTZ has also been proposed in the field of periodontal therapy either with a systemic administration or with local biodegradable sustained-release agents. The local administration of MTZ in the form of gel significantly reduces the systemic side effects. The aim of the present study, was to simulate the in vivo conditions occurring in diseased periodontal sites, and to evaluate the effects of MTZ on the viability of isolated HPLCs. The ability of MTZ to modulate the release of interleukin (IL)-1beta, IL-6, IL-8, IL-12 and tumor necrosis factor alpha (TNF-alpha) in HPLC, treated or not with LPS of Porphyromonas gingivalis was also evaluated. The results obtained showed that MTZ had no cytotoxic effect on HPLC and was able to inhibit the production of pro-inflammatory cytokines analyzed. The ability of MTZ to determine immunomodulatory effects could provide possible therapeutic applications in the field of periodontal research.

Effect of resveratrol and modulation of cytokine production on human periodontal ligament cells.

Periodontitis is a multifactorial polymicrobial infection characterized by a destructive inflammatory process. Porphyromonas gingivalis, a Gram-negative anaerobic black-pigmented rod, which produces several virulence factors that stimulate human periodontal ligament cells (HPLCs) to produce various inflammatory mediators, has been implicated as a crucial etiologic agent in the initiation and progression of periodontitis. Since natural polyphenols such as resveratrol have growth-inhibitory effects on some bacterial pathogens and have shown chemo-preventive, anti-inflammatory and antioxidant activity, in the present study we used an HPLC model stimulated with lipopolysaccharide (LPS) of P. gingivalis to simulate the in vivo conditions such as those found in diseased periodontal sites. To determine whether resveratrol interferes with P. gingivalis LPS-activity and reduces the production of pro-inflammatory molecules, we investigated its effect on the cytokines IL-1β, IL-6, IL-8, IL-12 and TNF-α and NO production of HPLCs. The results showed that resveratrol treatment decreased in a dose- and time-dependent manner the NO expression induced by P. gingivalis LPS, correlated to an increased viability of infected HPLCs, and decreased the production of pro-inflammatory cytokines in HPLCs stimulated by P. gingivalis LPS. These results suggest that the ability of resveratrol to determine immunomodulatory effects could provide possible therapeutic applications for the treatment of periodontitis.

Biological response of human bone marrow mesenchymal stem cells to fluoride‐modified titanium surfaces

OBJECTIVES The aim of the present study was to examine the behaviour of human bone marrow-derived mesenchymal stem cells (BM-MSC) to fluoride-modified grit-blasted (F-TiO) titanium surfaces compared with grit-blasted ones (TiO). MATERIAL AND METHODS Implant surfaces were analysed by atomic force microscopy (AFM) and scanning electron microscopy (SEM). BM-MSC were isolated from healthy donors and grown on the implant surfaces. Cell adhesion and proliferation, type I collagen (Col I) synthesis, osteoblastic differentiation (in terms of alkaline phosphatase activity, osteocalcin synthesis and extracellular matrix mineralization) were assessed. Furthermore, the ability to affect the osteoblastic/osteoclastic balance in terms of osteoprotegerin (OPG) and activator of nuclear factor κ B ligand (RANKL) ratio was investigated. RESULTS F-TiO surface showed higher S(a) values (P<0.05) and the presence of nano-scale structures at the AFM and SEM analysis. Comparable cell morphology and similar adhesion values on both surfaces were detected at early time, whereas higher proliferation values on F-TiO samples were observed at 7 and 10 days. Increased Col I and OPG levels for cells grown on F-TiO were found, whereas RANKL was not detectable in any of the conditioned media. BM-MSC showed a similar expression of early and late osteogenic markers on both TiO and F-TiO surfaces. CONCLUSIONS The results of the present study show that the chemical and micro/nano-scale modifications induced by fluoride treatment of TiO-grit blasted surfaces stimulate the proliferation and the extracellular matrix synthesis by BM-MSC, as well as the increase of OPG synthesis, thus preventing osteoclast activation and differentiation.

Human gingival fibroblast functions are stimulated by oxidized nano-structured titanium surfaces

OBJECTIVES The aim of this study was to analyze the features of an oxidized titanium implant surface and to evaluate its effects on the response of human gingival fibroblasts. METHODS 10mm×10mm×1mm turned (control) and oxidized (test) titanium samples (P.H.I. s.r.l., Italy) were examined by scanning electron microscopy and atomic force microscopy and characterized by height, spatial and hybrid roughness parameters. Primary cultures of human gingival fibroblasts were seeded on titanium samples, and cell morphology, adhesion, proliferation and extracellular matrix deposition, in terms of type I collagen synthesis, were evaluated. RESULTS Control and test surfaces appeared considerably different at the microscopic analyses: turned samples were grooved, whereas oxidized surfaces showed a more complex micro- and nano-scaled texture, as evidenced by roughness parameters. Cell adhesion and proliferation rate, as well as collagen synthesis, were greater on oxidized vs turned surfaces. CONCLUSIONS Although both control and test samples were in the range of average roughness proper of smooth surfaces, they exhibited significantly different topographic properties in terms of height and, mostly, hybrid parameters. Furthermore, oxidized surfaces enhanced human gingival fibroblast adhesion, proliferation and extracellular matrix deposition, and this could be due to the different structure at micro- and nano-scale levels. CLINICAL SIGNIFICANCE Oxidized nanostructured titanium surfaces could have a significant clinical utilization in virtue of their affinity for soft tissue attachment at the implant neck and/or at the transmucosal portion of the prosthetic abutment.

Bone marrow mesenchymal stem cell response to nano-structured oxidized and turned titanium surfaces.

OBJECTIVES The aim of this study was to analyse the topographic features of a novel nano-structured oxidized titanium implant surface and to evaluate its effect on the response of human bone marrow mesenchymal stem cells (BM-MSC) compared with a traditional turned surface. METHODS The 10 × 10 × 1 mm turned (control) and oxidized (test) titanium samples (P.H.I. s.r.l.) were examined by scanning electron microscopy (SEM) and atomic force microscopy (AFM) and characterized by height, spatial and hybrid roughness parameters at different dimensional ranges of analysis. Primary cultures of BM-MSC were seeded on titanium samples and cell morphology, adhesion, proliferation and osteogenic differentiation, in terms of alkaline phosphatase activity, osteocalcin synthesis and extracellular matrix mineralization, were evaluated. RESULTS At SEM and AFM analyses turned samples were grooved, whereas oxidized surfaces showed a more complex micro- and nano-scaled texture, with higher values of roughness parameters. Cell adhesion and osteogenic parameters were greater on oxidized (P<0.05 at least) vs. turned surfaces, whereas the cell proliferation rate was similar on both samples. CONCLUSIONS Although both control and test samples were in the range of average roughness proper of smooth surfaces, they exhibited significantly different topographic properties in terms of height, spatial and, mostly, of hybrid parameters. This different micro- and nano-structure resulted in an enhanced adhesion and differentiation of cells plated onto the oxidized surfaces.

Modulation of cytokine and β-defensin 2 expressions in human gingival fibroblasts infected with Chlamydia pneumoniae

Human beta-defensin 2 is an antimicrobial peptide that is produced by several epithelial cells after stimulation with micro-organisms and inflammatory mediators. Gram-negative bacteria, which are typically detected in periodontal pockets in periodontitis, elicit a stronger antibacterial peptide response of human beta-defensin 2 by epithelial cells. In this study, we investigated whether Chlamydia pneumoniae is able both to enter and grow in human gingival fibroblasts (HGF), to modify the production of cytokines, and is involved in regulation of beta-defensin 2 expression. Gingival fibroblasts discarded from periodontal procedures on healthy young individuals were infected with viable C. pneumoniae or with heat- or ultraviolet-inactivated organisms at a multiplicity of infection of 4 inclusion-forming units per cell. Our results demonstrate that after 48 h of incubation with viable C. pneumoniae, gingival fibroblasts showed a proliferative response as seen by both colorimetric assay and direct cell count (40% and 45%, respectively). Moreover, cells incubated with viable or ultraviolet light-inactivated C. pneumoniae organisms showed an increase in the levels of interleukin-6, interleukin-10 and human beta-defensin 2 in a time-dependent fashion, while the cells infected with heat-killed bacteria did not show a significant production either of the cytokines or beta-defensin 2 at any time. In conclusion, we demonstrate the correlation between multiplication of C. pneumoniae in human gingival fibroblasts and release of interleukin-6, interleukin-10 and up-regulation of beta-defensin 2, suggesting that gingival fibroblasts may be a periodontium niche for obligate intracellular C. pneumoniae and may play a role in innate gingival immune system and inflammatory response mechanisms of periodontitis.

Development and performance analysis of PCL/silica nanocomposites for bone regeneration

In the present article, several developments of biocomposites containing silica nanoparticles intended for bone regeneration are reported. Nanocomposites of poly(ε-caprolactone) (PCL) and silica, in which either the silica nanoparticles or the PCL have been modified in order to improve interfacial adhesion through chemical graft between the phases are hereafter described. The composites are characterized with respect to their chemical–physical and mechanical properties. Their biocompatibility and capacity to induce the osteoblastic phenotype in human bone marrow mesenchymal stem cells have been assessed.

Bacterial inactivation/sterilization by argon plasma treatment on contaminated titanium implant surfaces: In vitro study.

Background Surface treatment by argon plasma is widely used as the last step of the manufacturing process of titanium implant fixtures before their sterilization by gamma rays. The possibility of using such a technology in the daily clinical practice is particularly fascinating. The aim of the present study was to assess the effects of the argon plasma treatment on different titanium implant surfaces previously exposed In vitro to bacterial contamination. Material and Methods Sterile c.p. titanium implant discs with turned (T, Sa: 0.8 µm ), sandblasted/acid-etched (SAE, Sa: 1.3 µm) and titanium plasma sprayed (TPS, Sa: 3.0µm) surface were used in this study. A strain of Aggregatibacter actinomycetemcomitans ATCC3718 was grown at 37°C under anaerobic conditions for 24 h and then transferred on six discs for each of the three surface types. After 24 hours, a half of the contaminated discs (control group) were directly used to evaluate the colony forming units (CFUs). The other half of the contaminated discs (test group) were treated in an argon plasma chamber for 12 minutes at room temperature prior to be analyzed for CFU counting. All assays were performed using triplicate samples of each material in 3 different experiments. Results When the CFU counting was carried out on control discs, a total of 1.50x106±1.4x105, 1.55x106±7.07x104 and 3.15x106±2.12x105 CFU was respectively assessed for T, SAE and TPS discs, without statistically significant differences among the three surfaces. On the contrary, any trace of bacterial contamination was assessed for titanium discs treated in the argon plasma chamber prior to be analyzed, irrespectively to the implant surface tested. Conclusions Within the limit of this study, reported data suggested that the argon plasma technology could be efficiently used to decontaminate/sterilize previously infected titanium implant surfaces. Key words:Argon plasma, titanium implant surface, Aggregatibacter actinomycetemcomitans.

The Effect of Titanium Surface Modifications on Dental Implant Osseointegration.

The use of titanium dental implants has consistently changed the way of rehabilitating patients in modern dentistry and can count on high long-term survival and success rates. With respect to its introduction in the late 1960s, clinical indications for implant therapy have been significantly extended and optimal results have been achieved, even in clinical conditions formerly considered as unfavourable. Such evolution owes a lot to the significant progress made in the field of titanium surfaces. The topographical and chemical modification of traditional titanium surfaces has led to a real epochal shift in implant dentistry. Depending on the type and characteristics of the surface treatment applied, a wide range of implants has been produced, which have contributed to the success but also increased the risk of confusion in implant selection criteria for the clinician. This chapter, which provides an updated analysis of the relevant literature, the characteristics of modern implant surfaces, the biological principles underlying their role in promoting osseointegration and the scientific evidence about their clinical use are analyzed and presented.