Marcello Piccirilli

Regenerative potential of human periodontal ligament derived stem cells on three-dimensional biomaterials: A morphological report

Recent studies have shown that mesenchymal stem cells obtained from periodontal ligament (PDL-MSCs) are multipotent cells that have similar features of the bone marrow and dental pulp MSCs and are capable of proliferating and producing different types of tissue such as bone and tooth associated-tissues. Human PDL-MSCs expanded ex vivo were induced to osteogenesis, seeded in three-dimensional biocompatible scaffolds (fibrin sponge, bovine-derived substitutes) and examined using light, scanning and transmission electron microscopy. Morphological observations showed extensive growth of cellular biomass partially covering the scaffolds after 4 weeks of incubation in mineralization medium. These findings indicate that periodontal ligament can be an easily and efficient autologous source of stem cells with a high expansion capacity and ability to differentiate in osteogenic cells that can colonize and grow connected to bio-compatible scaffold. It can be suggested that the use of PDL-MSCs for generating graft biomaterials is advantageous for bone tissue engineering in regenerative dentistry.

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.

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.

VEGF and MVD expression in sinus augmentation with autologous bone and several graft materials

OBJECTIVE The aim of this study was to assess vascular endothelial growth factor (VEGF) expression and microvessel density (MVD) in maxillary sinus augmentation with autogenous bone and different graft materials for evaluating their angiogenic potential. METHODS Biopsies were harvested 10 months after sinus augmentation with a combination of autogenous bone and different graft materials: hydroxyapatite (HA, n = 6 patients), demineralized freeze-dried bone allograft (DFDBA, n = 5 patients), calcium phosphate (CP, n = 5 patients), Ricinus communis polymer (n = 5 patients) and control group--autogenous bone only (n = 13 patients). RESULTS In all the samples, higher intensities of VEGF expression were prevalent in the newly formed bone, while lower intensities of VEGF expression were predominant in the areas of mature bone. The highest intensity of VEGF expression in the newly formed bone was expressed by HA (P < 0.001) and CP in relation to control (P < 0.01) groups. The lowest intensities of VEGF expression in newly formed bone were shown by DFDBA and polymer groups (P < 0.05). When comparing the different grafting materials, higher MVD were found in the newly formed bone around control, HA and CP (P < 0.001). CONCLUSION Various graft materials could be successfully used for sinus floor augmentation; however, the interactions between bone formation and angiogenesis remain to be fully characterized.

Endomyocardial infiltration by B and NK cells foreshadows the recurrence of cardiac allograft rejection.

Heart allograft outcome is unpredictable and acute rejection episodes still occur despite the improvement of immunosuppressive regimens. We therefore investigated whether the immunopathological profile of endomyocardial biopsies might underlie the variations in the clinical course of a graft. Biopsies from transplanted patients were analysed by histology, immunohistochemistry (associated with digital image analysis), confocal and electron microscopy to determine the type and the functional state of leukocytes infiltrating the myocardium, together with their ultrastructural features and those of the graft itself. In comparison with biopsies with grade 0R or grade 1R rejection, those from patients with grade 2R rejection displayed significant infiltration of macrophages, T lymphocytes, and CD83+ and DC‐SIGN+ dendritic cells. Fifty‐seven per cent were invaded by CD20+B lymphocytes, most of which expressed CD69 activation marker and cooperated in interleukin‐12 production, and by CD69+CD94+NK cells expressing interferon‐γ. Ultrastructural signs of myocyte degeneration and microvessel rupture by NK cells were frequent. These patients developed recurrent episodes of acute allograft rejection. Endomyocardial B and NK infiltrates are involved in the dynamics of allograft rejection and are associated with a high risk of its recurrence. Immunopathological assessment of endomyocardial biopsies may thus serve to forecast the probable outcome of a heart allograft. Copyright

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.

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.

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.

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 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.