The integration of orthodontic miniscrews under mechanical loading: a pre-clinical study in rabbit.

INTRODUCTION: Orthodontic miniscrews are an increasingly popular choice to achieve absolute anchorage. The temporary use of miniscrews and their recent introduction have limited the debate over the biological aspect of the materials to that of the surface that permeates the field of dental implants. The aim of the present study was to investigate the integration of grade 5 titanium mini-implants with machined or sand blasted acid etched surface (SAE) under mechanical load in a rabbit tibia model of implant integration. METHODS: A total of 64 miniscrews (Ti6Al4V) of 1.5 mm diameter and 6.5 mm length were inserted in the proximal medial surface of each tibia in eight male rabbits aged 6 months. Each tibia received four miniscrews. A 100 g nickel-titanium coil spring (Neosentalloy) was applied between two miniscrews along the main axis while two miniscrews were left unloaded. The removal torque was measured for loaded and unloaded miniscrews after 12 weeks. Two miniscrews were harvested for histology. RESULTS: Removal torque was significantly higher for SAE mini-implants than for machined screws, under both loading conditions. Although no difference in bone to implant contact was observed among the groups, cortical area significantly decreased with both surfaces under loading. CONCLUSIONS: Our data indicate that SAE miniscrews have higher bone retention than MA miniscrews, although the effects of mechanical loading of these devices on cortical bone require further investigations.

Biomimetic coating with phosphoserine-tethered poly(epsilon-lysine) dendrons on titanium surfaces enhances Wnt and osteoblastic differentiation.

OBJECTIVES: Phosphoserine-based functionalization has been proposed as a tool to improve integration of endosseous implants by promoting osteoblast adhesion and differentiation in vitro. The present work investigates whether phosphoserine-tethered poly(epsilon-lysine) dendrons, when applied as a film to titanium surfaces, enhance the differentiation of osteoblastic cells and the activation of Wnt/ß-catenin signaling. MATERIALS AND METHODS: These films were tested in a murine model of calvaria-derived MC3T3 osteoblastic cells, primary bone marrow cells and mesenchymal, undifferentiated C2C12 cells. Gene expression was assayed by Real Time PCR, and activation of Wnt signaling pathway was measured with a reporter assay. RESULTS: Dendrons increased expression of alkaline phosphatase and osteocalcin, two osteoblastic markers, in both murine osteoblastic MC3T3 cells and primary bone marrow cells. The expression of osteoprotegerin, a protein opposing osteoclastogenesis was also significantly higher in cells growing on dendron-coated substrates both at 3 and 6 days of culture. Similarly, the mRNA levels of Wisp-2 and of ß-catenin, two Wnt target genes, were also markedly increased in this group at day 6. The activation of this signaling pathway in cells growing on the dendron-coated surfaces was confirmed by use of a TCF/ß-catenin reporter system in the C2C12 cell line. CONCLUSIONS: The findings of the present study show that phosphoserine-tethered poly(epsilon-lysine) dendron films act as stimuli for the activation of specific signal cascades and promote the differentiation of adhering progenitor cells into an osteoblastic phenotype.

Serotonin: a novel bone mass controller may have implications for alveolar bone.

As recent studies highlight the importance of alternative mechanisms in the control of bone turnover, new therapeutic approaches can be envisaged for bone diseases and periodontitis-induced bone loss. Recently, it has been shown that Fluoxetine and Venlafaxine, serotonin re-uptake inhibitors commonly used as antidepressants, can positively or negatively affect bone loss in rat models of induced periodontitis. Serotonin is a neurotransmitter that can be found within specific nuclei of the central nervous system, but can also be produced in the gut and be sequestered inside platelet granules. Although it is known to be mainly involved in the control of mood, sleep, and intestinal physiology, recent evidence has pointed at far reaching effects on bone metabolism, as a mediator of the effects of Lrp5, a membrane receptor commonly associated with Wnt canonical signaling and osteoblast differentiation. Deletion of Lrp5 in mice lead to increased expression of Tryptophan Hydroxylase 1, the gut isoform of the enzyme required for serotonin synthesis, thus increasing serum levels of serotonin. Serotonin, in turn, could bind to HTR1B receptors on osteoblasts and stop their proliferation by activating PKA and CREB.Although different groups have reported controversial results on the existence of an Lrp5-serotonin axis and the action of serotonin in bone remodeling, there is convincing evidence that serotonin modulators such as SSRIs can affect bone turnover. Consequently, the effects of this drug family on periodontal physiology should be thoroughly explored.

Effect of surface treatment on cell responses to grades 4 and 5 titanium for orthodontic mini-implants.

INTRODUCTION: Mini-implants are used to improve orthodontic anchorage, but optimal composition and surface characteristics have yet to be determined. We investigated the behavior of osteoblast-like cells on grade 4 commercially pure titanium and grade 5 titanium alloy with different surface treatments for mini-implants. METHODS: MC3T3 cells were plated on machined, acid-etched, or acid-etched grade 4 titanium enriched with calcium phosphate, or machined, anodized, or anodized and calcium phosphate-enriched grade 5 titanium disks. Surface and cell morphologies were assessed by scanning electron microscopy. Cell viability was measured by chemiluminescence, cytoskeletal organization was investigated by immunofluorescence, and real-time polymerase chain reaction for osteoblast-specific genes was performed to measure cell differentiation. RESULTS: Flattened shapes and strong stress fibers were observed on the machined surfaces; cells on the rough surfaces had a spindle shape, with lower cytoskeletal polarization. Cell proliferation was highest on smooth grade 4 titanium surfaces, whereas cells quickly reached a plateau on rough grade 4 titanium; no difference was observed after 72 hours in the grade 5 titanium groups. Calcium phosphate enrichment on grade 4 titanium significantly increased the messenger RNA levels for alkaline phosphatase and osteocalcin. Osteoblastic markers were higher on the grade 5 titanium machined surfaces than on the rough surfaces, and comparable with acid-etched grade 4 titanium. CONCLUSIONS: Although the grade 4 titanium enriched with calcium phosphate had the highest level of differentiation in vitro, the grade 5 titanium machined surfaces supported cell proliferation and matrix synthesis, and induced high expression of early differentiation markers. Increased mechanical resistance of grade 5 titanium makes it a potential candidate for orthodontic mini-implants.

Tolerable upper intake level for dietary sugars.

Following a request from five European Nordic countries, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was tasked to provide scientific advice on a tolerable upper intake level (UL) or a safe level of intake for dietary (total/added/free) sugars based on available data on chronic metabolic diseases, pregnancy-related endpoints and dental caries. Specific sugar types (fructose) and sources of sugars were also addressed. The intake of dietary sugars is a well-established hazard in relation to dental caries in humans. Based on a systematic review of the literature, prospective cohort studies do not support a positive relationship between the intake of dietary sugars, in isocaloric exchange with other macronutrients, and any of the chronic metabolic diseases or pregnancy-related endpoints assessed. Based on randomised control trials on surrogate disease endpoints, there is evidence for a positive and causal relationship between the intake of added/free sugars and risk of some chronic metabolic diseases: The level of certainty is moderate for obesity and dyslipidaemia (> 50-75% probability), low for non-alcoholic fatty liver disease and type 2 diabetes (> 15-50% probability) and very low for hypertension (0-15% probability). Health effects of added vs. free sugars could not be compared. A level of sugars intake at which the risk of dental caries/chronic metabolic diseases is not increased could not be identified over the range of observed intakes, and thus, a UL or a safe level of intake could not be set. Based on available data and related uncertainties, the intake of added and free sugars should be as low as possible in the context of a nutritionally adequate diet. Decreasing the intake of added and free sugars would decrease the intake of total sugars to a similar extent. This opinion can assist EU Member States in setting national goals/recommendations.

Adhesion pattern and growth of primary human osteoblastic cells on five commercially available titanium surfaces.

OBJECTIVE: The aim of this study is to analyze the morphology and proliferation of human osteoblastic cells in vitro on five commercially available titanium surfaces. MATERIALS AND METHODS: Human primary cells of the osteoblastic lineage were obtained from bone explants. The cells were plated on polished (T1), machined (T2), sand-blasted/acid-etched (T3), sand-blasted/acid-etched, modified with hydrogen peroxide rinse (T4), and plasma-sprayed titanium (T5) disks. Cell morphology was studied after 6, 24, 72 h, 7 and 14 days of culture by scanning electron microscopy. The formation and distribution of focal adhesions was investigated by immunocytochemical staining at 3, 6 and 24 h. Cell growth was measured by an MTT assay after 3, 7 and 9 days of culture. Moreover, the production of osteocalcin and osteoprotegerin (OPG) was evaluated in the supernatants by ELISA. RESULTS: Morphological analysis revealed that substrate topography profoundly affected cells' shape and their anchoring structures. Large lamellipodia were formed on polished and machined surfaces, while thin filopodia were more frequently observed on T3 and T4 samples. Moreover, cells formed stronger focal adhesions on T3 and T4 surfaces, and cell proliferation was higher on rough surfaces. Osteocalcin production was higher on the T4 surface, whereas OPG steadily increased on every surface. CONCLUSIONS: Taken together, these data show that all the surfaces allowed cell attachment, adhesion and proliferation, but T4 and T5 surfaces appeared to be a better substrate for the adhesion, proliferation and differentiation of cells of the osteoblastic lineage.

3D-printed chitosan scaffolds modified with D-(+) raffinose and enriched with type IV collagen to improve epithelial cell colonization.

Tissue regeneration often requires the use of biocompatible resorbable scaffolds to support the ingrowth of cells from neighboring tissues into a localized tissue defect. Such scaffolds must possess surface molecular cues that stimulate cells to populate the device, the first necessary condition for the formation of a healthy tissue. Chitosan is a natural polymer that has long been tested in biomedical applications because of its high biocompatibility, which can be further increased by modifying its formulation, e.g. adding D-(+) raffinose. We used this formulation in an ad hoc designed 3D printer to create regularly ordered scaffolds, which we then enriched with type IV collagen, an isoform of collagen that is exclusively found in basement membranes. Human epithelial A549 cells were then seeded on control scaffolds or on scaffolds coated with collagen, which was precipitated, or on scaffolds first collagenized and then exposed to either UVB or UVC radiation. Observations by the transmission light microscope, confocal microscope after staining with calcein-AM/propidium iodide, and by environmental scanning electron microscope revealed that collagen-enriched UV-treated scaffolds promoted the attachment of a higher number of cells, which covered a more extensive area of the scaffold, as also confirmed by alamar blue viability assay. Together these data confirm that coating 3D-printed scaffolds made of D-(+) raffinose-modified chitosan with type IV collagen and exposing them to UV light sensibly increases the cell compatibility of scaffolds, making them a better candidate to serve as a tool for the regeneration of epithelia.

The Use of Pulsed Electromagnetic Fields to Promote Bone Responses to Biomaterials In Vitro and In Vivo.

Implantable biomaterials are extensively used to promote bone regeneration or support endosseous prosthesis in orthopedics and dentistry. Their use, however, would benefit from additional strategies to improve bone responses. Pulsed Electromagnetic Fields (PEMFs) have long been known to act on osteoblasts and bone, affecting their metabolism, in spite of our poor understanding of the underlying mechanisms. Hence, we have the hypothesis that PEMFs may also ameliorate cell responses to biomaterials, improving their growth, differentiation, and the expression of a mature phenotype and therefore increasing the tissue integration of the implanted devices and their clinical success. A broad range of settings used for PEMFs stimulation still represents a hurdle to better define treatment protocols and extensive research is needed to overcome this issue. The present review includes studies that investigated the effects of PEMFs on the response of bone cells to different classes of biomaterials and the reports that focused on in vivo investigations of biomaterials implanted in bone.

Osteoblasts preferentially adhere to peaks on micro-structured titanium.

The aim of the study was to investigate cell adhesion to micro-structured titanium. Osteoblastic MC3T3 cells were cultured on smooth (P) or sand-blasted/acid-etched (SLA) titanium discs and were observed at scanning electron microscope/focused ion beam (SEM/FIB). Myosin II and actin microfilaments were labelled for epifluorescence microscopy. FIB revealed that cell adhesion initiated centrally and expanded to the cell periphery and that cells attached on the substrate by bridging over the titanium irregularities and adhering mostly on surface peaks. Gaps were visible between concave areas and cytoplasm and areas around ridges represented preferred attachment points for cells. A different myosin distribution was observed between samples and myosin inhibition affected cell responses. Taken together our data indicate that cells attach on micro-rough titanium by bridging over its irregularities. This is likely mediated by myosin II, whose distribution is altered in cells on SLA discs.

Osteoprotegerin and receptor activator of nuclear factor-kappa B ligand modulation by enamel matrix derivative in human alveolar osteoblasts.

BACKGROUND: Bone regeneration techniques increasingly rely on the use of exogenous molecules able to enhance tissue formation in pathologic and traumatic defects. An enamel matrix derivative (EMD) has been largely used to promote tooth ligament regeneration within periodontal pockets. Recent evidence suggests that EMD may contribute to inducing osteoblast growth and differentiation. We investigated the effects of EMD on growth and osteogenic marker modulation in human mandibular osteoblasts. METHODS: We focused our attention on cell growth by 3-(4,5-dimethyl[thiazol-2-yl]-3,5-diphery)tetradium bromide (MTT) assay, cell differentiation, mineralized nodule formation, and, in particular, the expression of receptor activator of nuclear factor-kappa B ligand (RANKL), the main osteoclast differentiation factor, and its decoy receptor, osteoprotegerin (OPG), by enzyme-linked immunosorbent assay. RESULTS: Cell growth was significantly increased by EMD. Similarly, a significantly higher quantity of OPG and a lower amount of RANKL were detectable in groups treated with 50 and 100 microg/ml at weeks 1, 2, and 3, and alkaline phosphatase activity and osteocalcin production were enhanced in cultures treated with 50 and 100 microg/ml at weeks 2 and 3. Mineralized nodules appeared bigger and more numerous in cultures treated with 50 and 100 microg/ml EMD. CONCLUSIONS: EMD was able to enhance osteoblast cell growth and the expression of markers of osteoblastic phenotype and differentiation. EMD also seemed able to create a favorable osteogenic microenvironment by reducing RANKL release and enhancing osteoblastic OPG production.

Immediate provisional restoration of postextraction implants for maxillary single-tooth replacement.

The timing of implant placement and loading following tooth extraction has recently undergone substantial reconsideration. The authors tested a protocol of immediate loading of single implants placed at the time of tooth extraction in a consecutive case series. Thirty-three patients received a single implant-supported crown to replace a maxillary anterior tooth at the time of extraction. Regular recalls were planned for the following 4 years. One implant did not integrate, and another became unstable secondary to facial trauma. Overall patient satisfaction and clinical and radiographic parameters were good.

Comparison of Environmental Scanning Electron Microscopy in Low Vacuum or wet mode for the investigation of cell biomaterial interactions.

AIM: The aim of the present study was to investigate the efficacy of environmental scanning electron microscopy (ESEM), in low vacuum mode (LV-ESEM) and in wet mode (wet-ESEM) in the assessment of cell-material interactions. METHODS: Mouse calvaria MC3T3 cells (ATCC) were seeded on commercially pure machined titanium discs of 10 mm diameter in Dulbecco modified MEM, 10% Fetal Bovine Serum, 1% Penicillin and Streptomycin and 1% Glutamine. Samples were then processed for microscope observation by rinse in Phosphate Buffer saline and fixation in 4.5% Glutaraldehyde. Samples were then rinsed in Sodium Cacodylate buffer and observed or dehydrated in alcohol prior to LV-ESEM observation. Fresh samples in 0.9% NaCl solution were observed in wet- ESEM. RESULTS: No significant loss of detail was observed when dehydrated or non dehydrated samples were analysed at LV-ESEM.The observation of fresh samples in wet-ESEM however proved difficult for the need to eliminate water which forms a layer covering the sample, thus hiding cell surface details. When reducing the vapor pressure in the chamber, the layer evaporated and NaCl immediately started to precipitate and cells collapsed, thus no further investigation was possible. CONCLUSIONS: The use of low vacuum-ESEM after cell fixation, but without dehydration or gold sputter coating proved a viable alternative to traditional high vacuum SEM observation.

Comparison of human mandibular osteoblasts grown on two commercially available titanium implant surfaces.

BACKGROUND: Surface characteristics play a major role in determining tissue response to implants and therefore their clinical outcome. The aim of the present study was to compare two commercially available titanium surfaces: plasma sprayed (TPS) and sand-blasted, acid-etched surface (SLA). METHODS: The surfaces were characterized by roughness testing, scanning electronic microscopy (SEM), Raman spectroscopy, and protein adsorption to determine their microtopographic and chemical properties. The effect of the surfaces on human mandibular osteoblasts was then studied in terms of cell morphology, adhesion, proliferation, and differentiation. Human osteoblasts from the mandible were cultured on these two surfaces and evaluated at 3, 6, 24, and 48 hours to determine cell attachment and morphology. Growth and differentiation kinetics were subsequently investigated by evaluating cell growth, alkaline phosphatase activity, osteocalcin and osteoprotegerin production at 7, 14, and 21 days. RESULTS: Although roughness was quite similar, the two surfaces presented strong differences in their topography, and cell morphology varied as a consequence. Osteoblasts on SLA appeared more elongated and spindle shaped than those on TPS, and their adhesion at 3 and 6 hours was weaker, but reached that of cells on TPS at hour 24. Cell proliferation was greater on SLA surfaces but differentiation parameters; i.e., alkaline phosphatase and osteocalcin, provided better results on TPS surfaces. Osteoprotegerin production was enhanced on TPS surfaces at days 14 and 21. CONCLUSION: Although cells grown on both surfaces exhibited good adhesion capabilities, a well-differentiated osteoblastic phenotype, and maintained a clear proliferation potential, our study suggests that plasma-sprayed treatment offers a better performance than SLA by creating, at least in the early phases, better conditions for tissue healing.

Different titanium surface treatment influences human mandibular osteoblast response.

BACKGROUND: Six titanium disks with six different surface treatments were examined: SS: smooth (polished) surface; TPS: plasma spray; C100: sand blasting by aluminum oxide (Al2O3) diameter 100 microm and acid etching; C150: sand blasting by Al2O3 diameter 150 microm and acid etching; B60: sand blasting by zirconium oxide (ZrO2) diameter 60 microm and acid etching; and B120: sand blasting by ZrO2 diameter 120 microm and acid etching. METHODS: The surface characteristics were determined by scanning electron microscopy (SEM) observation and a roughness tester. Raman spectroscopy was used to determine the presence of residual substances on the samples. Cells were seeded onto the disk and after 24 hours, 6 days, and 12 days were observed under SEM and growth curves generated with a cell counter. Some samples were used to determine alkaline phosphatase activity (ALP), using a colorimetric assay. RESULTS: SEM observation revealed drastic differences in surface microtopography, with a higher cell density on sand-blasted and acid-etched (SLA) samples than SS and TPS, and more regularly aligned cells on B60 and B120 surfaces than on the others. The growth curves showed a greater adhesion of cells on the etched/blasted surfaces compared to the SS and TPS surfaces. The number of cells increased on all the SLA samples, especially B60, throughout the experiment. At the same time, there was considerable ALP activity on the B60 sample, while it remained at extremely low levels on SS and TPS surfaces. Raman analyses revealed Al2O3 debris on C100 and C150, partly explaining the poorer performances of these two surface treatments, since this substance was shown to be toxic for cultured osteoblasts. CONCLUSIONS: Surface treatments influence the growth and the metabolic activity of cultured osteoblasts, and B60 seems to be the most favorable surface inducing a more pronounced proliferation of cells together with a high differentiation degree.

Fresh-frozen bone blocks for horizontal ridge augmentation in the upper maxilla: 6-month outcomes of a randomized controlled trial.

PURPOSE: This randomized controlled trial compared fresh-frozen versus autologous bone blocks for maxillary horizontal ridge augmentation in patients with Cawood and Howell class IV atrophies. MATERIALS AND METHODS: Twenty-four patients were allocated to the autologous and fresh-frozen groups in a 1:1 ratio. Patients underwent computed tomography scans 1 week and 6 months after surgery for graft volume and density analysis. Doxycycline was administered at day 120 and day 150 to label new bone formation. Biopsy for histologic and histomorphometric analyses was performed at reentry for implant insertion, 6 months after grafting. RESULTS: Fresh-frozen grafts had lower density than autologous bone. Autologous and fresh-frozen grafts lost, respectively, 25% and 52% of their initial volume (p = .0041). Histology revealed the presence of newly formed bone within both graft types, but clear signs of inflammation were present in fresh-frozen blocks. CONCLUSIONS: According to these 6-month results, autologous bone blocks are preferable to fresh-frozen bone grafts.

Correlation between density and resorption of fresh-frozen and autogenous bone grafts.

TRIAL DESIGN: This analysis compared the outcome of fresh-frozen versus autologous bone block grafts for horizontal ridge augmentation in patients with Cawood and Howell class IV atrophies. METHODS: Seventeen patients received autologous grafts and 21 patients received fresh-frozen bone grafts. Patients underwent CT scans 1 week and 6 months after surgery for graft volume and density analysis. RESULTS: Two autologous and 3 fresh-frozen grafts failed. Autologous and fresh-frozen grafts lost, respectively, 28% and 46% of their initial volume (P = 0.028). It is noteworthy that less dense fresh-frozen blocks lost more volume than denser grafts (61% versus 16%). CONCLUSIONS: According to these 6-month results, only denser fresh-frozen bone graft may be an acceptable alternative to autologous bone for horizontal ridge augmentation. Further studies are needed to investigate its behaviour at longer time points.

The effect of age, gender, and insertion site on marginal bone loss around endosseous implants: results from a 3-year trial with premium implant system.

OBJECTIVES: The goal of this study was to evaluate bone changes around endosseous implants in partially edentulous patients. MATERIALS AND METHODS: A total of 632 two-stage implants were placed in 252 patients. The implants had straight emergence profile, ZirTi surface, 3.3 to 5 mm diameter, and 8.5 to 13 mm length. Bone levels were assessed on orthopantomography immediately after surgery and after 36 months and marginal bone loss (MBL) was calculated from their difference. RESULTS: Cumulative survival rate was 98.73%. Overall MBL was 0.8 mm ± 0.03 (mean ± SEM). Higher MBL was observed around implants in the maxilla than in the mandible (P < 0.007). A relation between implant diameter and MBL (P < 0.0001) was observed in male and, more limitedly, female patients. Older patients had higher MBL in the maxilla, but not in the mandible (P < 0.0001). MBL progressively increased with age in male patients, but reached a peak already in the 50-60 years age group in the female subset (P < 0.001). CONCLUSIONS: The overall MBL is consistent with the available literature. Site difference and patient age and gender appear to significantly affect MBL, representing important factors to be considered during implant placement.

The effects of Er:YAG laser treatment on titanium surface profile and osteoblastic cell activity: an in vitro study.

BACKGROUND: Laser light has been proposed as a tool to decontaminate the surface of endosseous implants. The effects of this maneuver on the interactions between cells and surface, however, are poorly known. The goal of the present study is to investigate osteoblast growth and differentiation on three commercially available surfaces untreated or after irradiation by erbium-doped:yttrium, aluminum, and garnet (Er:YAG) laser at two levels: 150 and 200 mJ/pulse at 10 Hz. METHODS: Human osteoblastic Saos-2 cells were plated on machined, sandblasted and acid-etched titanium, or titanium plasma-sprayed disks. The effects of lasing were observed with a scanning electronic microscope, and cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Moreover, we measured the production of the osteoblast-specific protein osteocalcin and of osteoprotegerin in the supernatants by immunoenzymatic assays. RESULTS: Although no visible changes were observed on machined or titanium plasma-sprayed disk samples at the tested levels, titanium peaks on sandblasted and acid-etched titanium disks appeared fused as a consequence of laser irradiation. Interestingly, cell proliferation was slower on irradiated titanium at both intensities on all the surfaces. Cell differentiation, as assessed by osteocalcin production, was generally unaffected by laser treatment, whereas the production of osteoprotegerin was decreased on all the surfaces irradiated at the intensity of 200 mJ/10Hz. CONCLUSIONS: These results indicate that Er:YAG laser at energy levels used in this study can alter the surface profile of titanium implants and these changes may negatively affect the viability and the activity of osteoblastic cells. Therefore, Er:YAG lasers should be used with caution on titanium surfaces.

Effect of laser-induced dentin modifications on periodontal fibroblasts and osteoblasts: a new in vitro model.

BACKGROUND: The erbium-doped:yttrium, aluminum, and garnet (Er:YAG) laser has been shown to be a promising tool for root treatment in periodontitis, but little information is available regarding the surface characteristics after this treatment, mainly because it is difficult to obtain standardized dentin samples for in vitro studies. METHODS: Commercially available standardized dentin disks were treated with an Er:YAG laser at different settings and used as a substrate for human primary osteoblastic cells (hOBs) and periodontal ligament fibroblasts (PLFs). Cell proliferation on untreated dentin was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after 3, 6, 12, 24, and 48 hours of culture. The effects of the laser on dentin and cell morphology on treated and untreated samples were investigated by scanning electron microscopy after 3, 6, 24, and 48 hours of culture. RESULTS: Dentin samples supported proliferation for both cell types, although growth kinetics were different. The laser dramatically affected the dentin profile, creating a rough and irregular surface. Cells grew easily on untreated dentin, but fewer cells were present on treated areas, often displaying long filopodes. hOBs showed poorer adhesion to treated dentin than PLFs. CONCLUSIONS: The dentin disks provide a standardized and useful tool to study dentin surface modifications in vitro. PLFs behaved differently from hOBs on dentin, possibly because of their different affinity to this tissue and/or their differentiation state. The changes induced by the laser produced a less favorable environment for cell adhesion or growth, and treated dentin seemed to be more suitable for PLF adhesion compared to hOB adhesion.