Nuclear fractal dimension in oral squamous cell carcinoma: a novel method for the evaluation of grading, staging, and survival.

Fractal dimension (FD) in tissue specimens from patients with oral squamous cell carcinoma (OSCC) was evaluated. FD values in different stages of OSCC, and the correlations with clinicopathological variables and patient survival were investigated. Histological sections from OSCC and control non-neoplastic mucosa specimens were stained with hematoxylin-eosin for pathological analysis and with Feulgen for nuclear evaluation. FD in OSCC groups vs. controls revealed statistically significant differences (P < 0.001). In addition, a progressive increase of FD from stage I and II lesions and stage III and IV lesions was observed, with statistically significant differences (P = 0.003). Moreover, different degrees of tumor differentiation showed a significant difference in the average nuclear FD values (P = 0.001). A relationship between FD and patients' survival was also detected with lower FD values associated to longer survival time and higher FD values with shorter survival time (P = 0.034). These data showed that FD significantly increased during OSCC progression. Thus, FD could represent a novel prognostic tool for OSCC, as FD values significantly correlated with patient survival. Fractal geometry could give insights into tumor morphology and could become an useful tool for analyzing irregular tumor growth patterns.

Matrix metalloproteinases 2, 3, 8, 9, and 13 in the peri-implant soft tissues around titanium and zirconium oxide healing caps.

PURPOSE: This human study sought to compare, from an immunohistochemical point of view, matrix metalloproteinases (MMPs) 2, 3, 8, 9, and 13 in the soft tissues around titanium and zirconium oxide healing caps. MATERIALS AND METHODS: Five patients participated in this study. All patients received 3.8 × 11-mm dental implants, which were left to heal in a nonsubmerged (single-stage) mode. Healing caps (3.8 mm in diameter and 3.0 mm in height) were inserted in all implants. Half of the implants were randomly supplied with standard, prefabricated caps of commercially pure titanium (control), while the other half were randomly provided with zirconium oxide caps (test). After a 6-month healing period, gingival biopsy specimens were obtained with a circular scalpel around the healing caps of both groups, without unscrewing or removing the healing caps, and the samples underwent immunohistochemical processing for MMPs 2, 3, 8, 9, and 13. RESULTS: Statistically significant differences were found in the values of MMP-8 in the cells of the inflammatory infiltrate, with higher values for the titanium samples. Statistically significantly higher values were found, also in the titanium samples, for MMP-9 in the endothelial cells of the blood vessels. No statistically significant differences were found for any other MMPs. CONCLUSIONS: The present results showed that the soft tissues around titanium healing caps underwent a higher rate of restorative processes, most probably correlated to the MMP levels observed in the tissues.

Histological and immunohistochemical evaluation of the peri-implant soft tissues around machined and acid-etched titanium healing abutments: a prospective randomised study.

A close spatial correlation has been described between the roughness of intraoral materials and the rate of bacterial colonisation. The aim of the present study in man was to conduct a comparative immunohistochemical evaluation of the inflammatory infiltrate, microvessel density, the nitric oxide synthases 1 and 3 and the vascular endothelial growth factor expression, the proliferative activity, and the B and T lymphocyte and histiocyte positivity in the peri-implant soft tissues around machined and acid-etched titanium healing caps. Ten patients participated in this study. The patients were enrolled consecutively. All patients received dental implants left to heal in a non-submerged mode. Healing caps were inserted in all implants. Half of the implants were supplied randomly with machined caps of titanium (control), while the other half were provided randomly with acid-etched titanium caps (test). After a 6-month healing period, a gingival biopsy was performed with a circular scalpel around the healing caps of both groups. The inflammatory infiltrate was mostly present in test specimens. Their extension was much larger than that of the control samples. A higher number of T and B lymphocytes were observed in test specimens. Higher values of microvessel density and a higher expression of vascular endothelial growth factor intensity were observed in the test samples. Furthermore, the Ki-67, NOS1 and NOS3 expression was significantly higher in the test specimens. All these results showed that the tissues around test healing caps underwent a higher rate of restorative processes, most probably correlated to the higher inflammation processes observed in these tissues.

Sinus lift with autologous bone alone or in addition to equine bone: an immunohistochemical study in man.

PURPOSE: The aim of the present study was to perform a comparative histological and immunohistochemical evaluation of microvessel density, vascular endothelial growth factor, and nitric oxide synthase (NOS) expression in sinus augmentation using autologous bone alone or in combination with equine bone. MATERIALS AND METHODS: Sixteen patients underwent sinus augmentation with autologous bone and a 50:50 mixture of autologous and equine bone. Control cores were harvested from preexisting nonaugmented bone under the sinus floor. The specimens were processed for immunohistochemistry. RESULTS: The greatest values of microvessel density were found in the sites grafted with autologous bone with significant differences between control versus autologous group (P < 0.01) and control versus autologous + equine group (P < 0.01). The higher and lower intensities of vascular endothelial growth factor and NOS3 expression were prevalent in the sites grafted with autologous bone with significant differences with the controls (P < 0.05). No significant differences (P > 0.05) were found in the NOS1 expression among the groups. CONCLUSION: The results obtained showed that the mixture of autologous and equine bone was biocompatible, and its use was associated with new blood vessels ingrowth during healing, which has been found to be extremely important for bone formation.

Immunohistochemical analysis of inflammatory infiltrate in aggressive and chronic periodontitis: a comparative study.

This immunohistochemical study evaluated the inflammatory infiltrate with its cluster differentiation markers (CD 4, CD 8, CD 20, and CD 68) in aggressive and chronic periodontitis gingival tissues in order to identify the specific cell distribution. Twenty-seven human gingival biopsies were obtained and analyzed. Fourteen patients were suffering from chronic periodontitis and six from aggressive periodontitis; seven patients with healthy gingiva were included as the control group. The specimens were immunohistochemically stained for anti-CD 4 (T helper cells), anti-CD 8 (T cytotoxic/suppressor), anti CD-20 (B plasma cells) and anti CD-68 (macrophages). Chronic periodontitis samples were mainly dominated by CD 4 and CD 8+ cells. On the contrary, in aggressive periodontitis patients all four cell types (CD 4, CD 8, CD 20 and CD 68 + cells, respectively) were remarkably increased. CD 20+ cells were significantly (p < 0.05) more prevalent in aggressive versus chronic periodontitis. The control samples expressed lower CD 4, CD 8, CD 20 and CD 68+ cells confirming a none inflammatory state. The present study demonstrates prevalence for CD 20+ cells in aggressive periodontitis lesions. However, further studies need to be performed to confirm and identify a clear pattern of inflammatory cells and hereafter the mechanisms sustaining the disease.

Immunohistochemical expression of vascular endothelial growth factor (VEGF) in different types of odontogenic cysts.

The objective of the present study was to evaluate vascular endothelial growth factor (VEGF) expression in different types of odontogenic cysts. A total of 25 parakeratotic odontogenic keratocysts (POKCs), 16 orthokeratotic odontogenic keratocysts (OOKCs), and 28 follicular cysts (FCs) were evaluated semiquantitatively for immunohistochemical analysis of VEGF in epithelial cells, endothelial cells of blood vessels, inflammatory cells and focally stromal cells. A significant different expression of VEGF in all cell components was found in keratocysts compared to FCs. The POKCs (80%) and OOKCs (68%) showed more than 50% VEGF positive epithelial cells, whereas the majority of FCs (71%) were either negative in the epithelium or showed less than 10% positive cells. Similarly, the POKCs (88%) and OOKCs (68%) showed more than 50% positive endothelial cells, whereas the FCs (75%) were either negative or showed less than 10% VEGF positive endothelial cells. The highest percentage of cases with score 2 positivity in the stromal cells was observed in POKCs (68%); OOKCs showed a score 2 positivity in 44%, score 1 in 31% and score 0 in 25%, whereas 68% of FCs showed a score 0, 25% a score 1 and only 7% of cases showed a score 2. No statistically significant differences were observed between POKCs and OOKCs in VEGF expression in the epithelial and endothelial cells, whereas the positivity score in stromal cells was significantly higher in POKCs compared to OOKCs. The present results can support the hypothesis that angiogenesis is an active mechanism in the invasive growth of the OKC.

Inflammatory infiltrate, microvessel density, vascular endothelial growth factor, nitric oxide synthase, and proliferative activity in soft tissues below intraorally welded titanium bars.

BACKGROUND: The aim of the present study is a comparative evaluation of inflammatory infiltrate, microvessel density, vascular endothelial growth factor, nitric oxide synthase, and proliferative activity in soft tissues below intraorally welded titanium bars. METHODS: Twenty-two patients participated in this study. All patients carried immediately loaded one-stage titanium implants splinted with intraorally welded titanium bars. Each patient underwent two gingival biopsies, a control biopsy harvested from an area of mucosa 5 mm away from the titanium bar and a test biopsy from the mucosa below the titanium bar, which were histologically and immunohistochemically processed. RESULTS: No fractures or radiographically detectable alterations of the welded frameworks were present. In all the cases examined, the average of the modified plaque index was 1, no suppuration or bleeding on probing was present, and probing depth was < or =3 mm. However, the immunohistochemical analysis revealed some differences. The inflammatory infiltrate was mostly present in test sites and its extension was much larger than in control sites. Statistically significant differences were found in microvessel density and Ki-67 expression among control and test groups (P <0.0001). The high intensity of vascular endothelial growth factor, nitric oxide synthase 1, and nitric oxide synthase 3 expression were mainly detected in the test group, whereas the low intensities were mostly expressed in controls, with statistically significant differences (P <0.0001). CONCLUSIONS: In the present study, the immunohistochemical analysis shows that the tissues below the titanium bars underwent a higher rate of inflammatory and reparative processes. However, further long-term studies, where clinical and immunohistochemical data are collected in parallel, should be conducted for a better understanding of the expression pattern of inflammation markers.

Alveolar ridge regeneration with equine spongy bone: a clinical, histological, and immunohistochemical case series.

BACKGROUND: In the case of localized ridge atrophy, a ridge augmentation procedure, with the use of bone substitutes and barrier membranes, may then be necessary. PURPOSE: The aim of the present study was a clinical, histological, and immunohistochemical evaluation of an equine spongy bone in alveolar ridge augmentation procedures. MATERIALS AND METHODS: Five patients showing horizontal mandibular ridge defects participated in this study. A ridge augmentation was performed through an onlay apposition of equine bone covered by a titanium-reinforced membrane. After 6 months of healing, five bone cores from nonaugmented sites (control) and five from augmented sites (test) were retrieved. RESULTS: In test sites, no postoperative complications occurred. Horizontal bone width increased from 24 to 37 mm. In control sites, the newly formed bone represented 33%, and in test sites, 35% of the total area. The mean value of the microvessel density was 25.6 +/- 3.425 per mm(2) in controls, while 33.3 +/- 2.5 vessels per mm(2) in the test sites were found (p < .05). Both groups showed a high intensity (++) of vascular endothelial growth factor expression in the newly formed bone, while a low intensity (+) was found in the mature bone. CONCLUSION: Equine bone appeared to be biocompatible and to be associated with new vessel ingrowth. Within the limits of the small sample size, the present study indicated that equine bone could be used in mandibular ridge augmentations.

P16 expression in odontogenic tumors.

AIMS AND BACKGROUND: The aim of the study was to examine the immunohistochemical expression of a cell-cycle-related factor (p16) in order to elucidate its role in the growth and diffusion of odontogenic tumors. STUDY DESIGN: Thirty-six odontogenic tumors were divided into two groups according to their clinical behavior: group A and group B composed of tumors at low and high risk of recurrences, respectively. The ANOVA test was used to detect differences between the two groups. RESULTS: p16 was expressed in both groups, but with different localization. A statistically significant difference was found in p16 expression of peripheral cells, with an increase in the expression in group B compared to group A (P < 0.05). In addition, there was no significant difference in p16 positive expression of the central cells of odontogenic tumors, which was high in both groups. CONCLUSIONS: The present data show a correlation between p16 expression and the biological behavior of odontogenic tumors.

Loss of expression of TGF-beta1, TbetaRI, and TbetaRII correlates with differentiation in human oral squamous cell carcinomas.

Despite advances in biological and molecular characteristics, the prognosis of oral squamous cell carcinomas is still very unfavourable and is based on the classical clinicopathological parameters. However, tumors with similar clinicopathological characteristics may differ dramatically in their clinical outcome. Thus, the identification of novel prognostic factors is necessary to improve prognostic and therapeutic approaches. Transforming growth factor-beta1 (TGF-beta1) is a potent growth inhibitor of epithelial cell proliferation, thus, inactivation of TGF-beta1 signalling may play a role in cancer. The expression levels of TGF-beta1 and its type I and type II receptors (TbetaRI and TbetaRII) were assessed by immunohistochemical and Western blot analyses in 22 oral squamous cell carcinoma lesions, in their normal adjacent mucosa and in the squamous carcinoma cell lines FaDu and CAL27. Immunohistochemistry on 22 oral carcinomas and case-matched normal oral mucosae demonstrated that TGF-beta1, TbetaRI, and TbetaRII were intensively and homogeneously expressed in all normal epithelia. In contrast, TGF-beta1 and its receptors were significantly reduced in poorly (G3) differentiated tumors as compared to moderately (G2) and well differentiated (G1) lesions (p=2.8 x 10(-3), p=1.3 x 10(-3), p=2.8 x 10(-3) and p=1.3 x 10(-3), respectively). The progressive reduction of the expression levels was confirmed by Western blotting. The oral squamous carcinoma cell lines Cal27 and FaDu demonstrated a reduced and a lack of TbetaRI expression, respectively. A significant decrease of TbetaRII expression, as compared to Cal27 cells, was shown in FaDu cells. Thus, the decreased expression of TbetaRII combined with the absence of TbetaRI could account for the resistance of FaDu cells to the growth-inhibiting effect of TGF-beta1. TGF-beta1 and TGF-beta1 receptor expression significantly decreased as tumors became less differentiated and thus more aggressive, suggesting a functional role of these molecules in oral tumor progression.

Tissue healing under provisional restorations with ovate pontics: a pilot human histological study.

STATEMENT OF PROBLEM: Ovate pontics mimic the natural tooth contour and provide an esthetic result however, few studies have evaluated the histological changes in underlying tissues. PURPOSE: The purpose of this pilot study was to histologically evaluate the healing of gingival tissues in contact with provisional ovate pontics after 2 weeks. MATERIAL AND METHODS: Three patients requiring fixed partial dentures participated in this study. The provisional restorations consisted of a fixed partial denture having 2 ovate pontics: one in acrylic resin (Jet), with an ovate shell made of low-fusing ceramic (Duceram LFC); the other made completely of the same acrylic resin (control). After 2 weeks, biopsies of the gingival tissues beneath the pontics were retrieved for histological examination, and immunohistochemistry for evaluation of the vascular endothelial growth factor (VEGF) was performed. Data were analyzed using descriptive statistics. RESULTS: Clinically, all of the ovate pontic-prepared sites showed partial healing. Histologically, the thickness of the entire mucosa was similar in both specimens; however, in some regions, the epithelium presented ulcerations that were generally deeper and more frequent in control sites than in test sites. Immunohistochemical results showed that tissues beneath LFC pontics seemed to be less inflamed since they demonstrated a lower expression of VEGF (10.7 +/- .8) compared to those beneath acrylic resin ovate pontics (33.9 +/- 2.5). CONCLUSION: This pilot study demonstrated that the placement of provisional LFC ovate pontics may be advantageous for the reparative processes of the underlying tissues.

Maxillary sinus augmentation using an engineered porous hydroxyapatite: a clinical, histological, and transmission electron microscopy study in man.

Porous hydroxyapatite (HA) is a calcium-phosphate-based material that is biocompatible, nonimmunological, and osteoconductive, and has a macroporosity of about 200 to 800 microm. The pores seem to be able to induce migration, adhesion, and proliferation of osteoblasts inside the pore network and to promote angiogenesis inside the pore system. The aim of this study was to evaluate the clinical behavior and the histological and ultrastructural aspects of porous HA in maxillary sinus augmentation procedures. Twenty-four patients (19 men, 5 women; average age 53.4 years) in good general physical and mental health and with partially or completely edentulous maxillae were selected for this study. Six months after sinus floor elevation, at the time of dental implant placement, biopsies were carried out under local anesthesia. These bone cores were cut in half and were processed for light and transmission electron microscopy. After a mean 3 years after implantation, all implants are clinically in function and no surgical or prosthetic complications have occurred. Under light microscopy, newly formed bone was 38.5% +/- 4.5%, whereas the residual biomaterial represented 12% +/- 2.3% and the marrow spaces represented 44.6% +/- 4.2%. In addition, in the majority of cases, the biomaterial particles were in close contact with the bone, which appeared compact with the characteristic features of well-organized lamellar bone. A cement-like line was slightly visible at the bone-biomaterial interface, but there were no gaps or interposed connective tissue in between. A high quantity (about 40%) of newly formed bone was present. Bone was closely apposed to the biomaterials particles as shown in light microscopy and transmission electron microscopy. Moreover, no signs of inflammatory cell infiltrate or foreign body reaction were present. Also, most of the biomaterial was resorbed and only a small quantity (a little more than 10%) was still present. The results of our study show that porous HA can be a suitable synthetic material for bone regeneration in maxillary sinus augmentation procedures.

Screw vs cement-implant-retained restorations: an experimental study in the beagle. Part 2. Immunohistochemical evaluation of the peri-implant tissues.

Crestal bone loss has been reported to occur around dental implants. Even if the causes of this bone loss are not completely understood, the presence of a microgap between implant and abutment with a possible contamination of the internal portion of the implants has been suggested. The aim of this study was to see if there were differences in the vascular endothelial growth factor (VEGF) expression, microvessel density (MVD), proliferative activity (MIB-1), and inflammatory infiltrate in the soft tissues around implants with screwed and cemented abutments. Sandblasted and acid-etched implants were inserted in the mandibles of 6 Beagle dogs. Ten 3.5- x 10-mm root-form implants were inserted in each mandible. A total of 60 implants (30 with screwed abutments and 30 with cemented abutments) were used. After 12 months, all the bridges were removed and all abutments were checked for mobility. A total of 8 loosened screws (27%) were found in the screwed abutments, whereas no loosening was observed in cemented abutments. A gingival biopsy was performed in 8 implants with cemented abutments, in 8 implants with screwed abutments, and in 8 implants with unscrewed abutments. No statistically significant differences were found in the inflammatory infiltrate and in the MIB-1 among the different groups. No statistically significant difference was found in the MVD between screwed and cemented abutments (P = .2111), whereas there was a statistically significant difference in MVD between screwed and unscrewed abutments (P = .0277) and between cemented and unscrewed abutments (P = .0431). A low intensity of VEGF was prevalent in screwed and in cemented abutments, whereas a high intensity of VEGF was prevalent in unscrewed abutments. These facts could be explained by the effects induced, in the abutments that underwent a screw loosening, by the presence of bacteria inside the hollow portion of the implants or by enhanced reparative processes.

Histologic and ultrastructural analysis of regenerated bone in maxillary sinus augmentation using a porcine bone-derived biomaterial.

BACKGROUND: The purpose of the present study was the histologic and ultrastructural evaluation of a biomaterial composed of cortical pig bone in the form of granules. METHODS: After maxillary sinus augmentation using this biomaterial, 10 specimens were retrieved after 5 months in 10 patients using this biomaterial. The specimens were processed to be observed under light microscopy (LM) and transmission electron microscopy (TEM). Histomorphometric measurements were presented by means +/- standard deviations. RESULTS: LM showed that most of the particles were surrounded by newly formed bone. In some areas, the osteoid matrix was present; however, mainly compact bone was present at the interface. There was no evidence of an acute inflammatory infiltrate. The newly formed bone was 36% +/- 2.8% and marrow spaces were 38% +/- 1.6%, whereas residual grafted material was 31% +/- 1.6%. Under TEM, all phases of bone formation (osteoid matrix, woven, and lamellar bone) were observed in proximity with the biomaterial particles. The bone-biomaterial interface showed a close contact between the porcine bone particles and the surrounding bone that had mainly features of mature bone with numerous osteocytes. A lamina limitans was sometimes present at this interface. CONCLUSIONS: According to our knowledge, this is the first study presenting data on TEM of a porcine bone-derived biomaterial used in sinus augmentation procedures in humans. Our findings show that this is a biocompatible biomaterial that can be used for maxillary sinus augmentation procedures without interfering with the normal reparative bone processes.

Maxillary sinus augmentation with Bio-Oss particles: a light, scanning, and transmission electron microscopy study in man.

Biological interactions occurring at the bone-biomaterial interface are critical for long-term clinical success. Bio-Oss is a deproteinized, sterilized bovine bone that has been extensively used in bone regeneration procedures. The aim of the present study was a comparative light, scanning, and electron microscopy evaluation of the interface between Bio-Oss and bone in specimens retrieved after sinus augmentation procedures. Under light microscopy, most of the particles were surrounded by newly formed bone, while in a few cases, at the interface of some particles it was possible to observe marrow spaces and biological fluids. Under scanning electron microscopy, in most cases, the particle perimeter appeared lined by bone that was tightly adherent to the biomaterial surface. Transmission electron microscopy showed that the bone tissue around the biomaterial showed all the phases of the bone healing process. In some areas, randomly organized collagen fibers were present, while in other areas, newly formed compact bone was present. In the first bone lamella collagen fibers contacting the Bio-Oss surface were oriented at 243.73 +/- 7.12 degrees (mean +/- SD), while in the rest of the lamella they were oriented at 288.05 +/- 4.86 degrees (mean +/- SD) with a statistically significant difference of 44.32 degrees (p < 0.001). In the same areas the intensity of gray value was 172.56 +/- 18.15 (mean +/- SD) near the biomaterial surface and 158.71 +/- 21.95 (mean +/- SD) in the other part of the lamella with an unstatistically significant difference of 13.79 (p = 0.071). At the bone-biomaterial interface there was also an electron-dense layer similar to cement lines. This layer had a variable morphology being, in some areas, a thin line, and in other areas, a thick irregular band. The analyses showed that Bio-Oss particles do not interfere with the normal osseous healing process after sinus lift procedures and promote new bone formation. In conclusion, this study serves as a better understanding of the morphologic characteristics of Bio-Oss and its interaction with the surrounding tissues.