Edoardo Manfredi

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.

The response of osteoblastic MC3T3-E1 cells to micro- and nano-textured, hydrophilic and bioactive titanium surfaces

The aim of the present work was to investigate the morphology and activity of the murine osteoblastic cell line MC3T3 on control smooth (Machined), commercially available rough (ZT) titanium discs, and on titanium samples obtained by modifying the ZT treatment protocol, and herein labelled as ZTF, ZTM and ZTFM. Cells were evaluated at SEM and immunofluorescence for morphology and cell-to-cell interactions and by MTT assay and real time PCR for cell growth and function. Microscopy showed that ZT modified protocols could differently affect cell shape and distribution. All the tested surfaces showed good biocompatibility by viability assay. However, cells on smoother surfaces appeared to express higher levels of transcript for Collagen 1a1, the main component of extracellular matrix, by real time PCR. Expression of the early differentiation marker Alkaline Phosphatase was higher on ZTF surfaces and ZTM enhanced the expression of later osteoblastic markers Osteoprotegerin and Osteocalcin. Noteworthy, the expression of Connexin 43, a component of cell-to-cell contacts and hemichannels, followed a similar pattern to differentiation marker genes and was higher in cells on ZTM surfaces, consistently with the microscopic observation of cell clusters. Taken together, this data showed that ZTF and ZTM treatment protocols appeared to improve the basal sand-blasting/acid-etching ZT procedure with ZTM surfaces promoting the most mature stage of differentiation.

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.

Chitosan scaffold modified with D-(+) raffinose and enriched with thiol-modified gelatin for improved osteoblast adhesion.

The aim of the present study was to investigate whether chitosan-based scaffolds modified with D-(+) raffinose and enriched with thiol-modified gelatin could selectively improve osteoblast adhesion and proliferation. 2, 3 and 4.5% chitosan films were prepared. Chitosan suitability for tissue engineering was confirmed by protein adsorption assay. Scaffolds were incubated with a 2.5 mg ml(-1) BSA solution and the decrease of protein content in the supernatants was measured by spectrophotometry. Chitosan films were then enriched with thiol-modified gelatin and their ability to bind BSA was also measured. Then, 2% chitosan discs with or without thiol-modified gelatin were used as culture substrates for MC3T3-E1 cells. After 72 h cells were stained with trypan blue or with calcein AM and propidium iodide for morphology, viability and proliferation assays. Moreover, cell viability was measured at 48, 72, 96 and 168 h to obtain a growth curve. Chitosan films efficiently bound and retained BSA proportionally to the concentration of chitosan discs. The amount of protein retained was higher on chitosan enriched with thiol-modified gelatin. Moreover, chitosan discs allowed the adhesion and the viability of cells, but inhibited their proliferation. The functionalization of chitosan with thiol-modified gelatin enhanced cell spreading and proliferation. Our data confirm that chitosan is a suitable material for tissue engineering. Moreover, our data show that the enrichment of chitosan with thiol-modified gelatin enhances its biological properties.

Chitosan-based scaffold modified with D-(+) raffinose for cartilage repair: an in vivo study

BackgroundOsteochondral defects significantly affect patients’ quality of life and represent challenging tissue lesions, because of the poor regenerative capacity of cartilage. Tissue engineering has long sought to promote cartilage repair, by employing artificial scaffolds to enhance cell capacity to deposit new cartilage. An ideal biomaterial should closely mimic the natural environment of the tissue, to promote scaffold colonization, cell differentiation and the maintenance of a differentiated cellular phenotype. The present study evaluated chitosan scaffolds enriched with D-(+) raffinose in osteochondral defects in rabbits. Cartilage defects were created in distal femurs, both on the condyle and on the trochlea, and were left untreated or received a chitosan scaffold. The animals were sacrificed after 2 or 4 weeks, and samples were analysed microscopically.ResultsThe retrieved implants were surrounded by a fibrous capsule and contained a noticeable inflammatory infiltrate. No hyaline cartilage was formed in the defects. Although defect closure reached approximately 100% in the control group after 4 weeks, defects did not completely heal when filled with chitosan. In these samples, the lesion contained granulation tissue at 2 weeks, which was then replaced by fibrous connective tissue by week 4. Noteworthy, chitosan never appeared to be integrated in the surrounding cartilage.ConclusionsIn conclusion, the present study highlights the limits of D-(+) raffinose-enriched chitosan for cartilage regeneration and offers useful information for further development of this material for tissue repair.

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.

Combined photographic and ultrasonographic measurement of the ANB angle: a pilot study

ObjectiveThis study was performed to evaluate the feasibility of noninvasive measurement of the ANB angle using photographic and ultrasonographic methods.MethodsTwenty consecutive orthodontic patients were evaluated. The ANB angle and soft tissue thickness covering the N, A, and B cephalometric points were measured by lateral teleradiography; these measurements were made by two expert operators. The soft tissue thickness covering the N, A, and B cephalometric points was measured by ultrasonography; these measurements were also made by two expert operators. On a 1:1 photographic profile print on which the ultrasonographic points were marked, the ANB ultrasonographic angle was measured. The following comparisons were considered: averaged and single measurements of N, A, and B points by first versus second ultrasonographer; averaged and single ultrasonographic versus radiographic soft tissue thickness covering the N, A, B points; and averaged and single ultrasonographic versus radiographic measurements of ANB angle.ResultsHigh correlation and concordance of the averaged and single measurements, but no significant difference, was found between the two ultrasonographers. No statistically significant difference was found between the two methods for measuring averaged soft tissue thickness, but a 20% difference was found for the single measurements. High correlation and concordance between the ultrasonographic and radiographic measurements, but no significant difference, was found between the single and averaged ANB angle measurements.ConclusionUltrasonography seems to be a noninvasive and reliable technique for measurement of the ANB angle and may replace radiographic measurement in some cases.

Anti-fibronectin aptamers improve the colonization of chitosan films modified with D-(+) Raffinose by murine osteoblastic cells

The aim of the present study was to investigate how the enrichment of chitosan films with anti-fibronectin aptamers could enhance scaffold colonization by osteoblasts, by improving their adhesion and accelerating their proliferation. Chitosan discs were enriched with excess of anti-fibronectin aptamer. Aptamer adsorption on chitosan was monitored by measuring aptamer concentration in the supernatant by spectrophotometry, as well as its release, while functionalization was confirmed by labelling aptamers with a DNA intercalating dye. Chitosan samples were then characterized morphologically with atomic force microscopy and physically with contact angle measurement. Chitosan enrichment with fibronectin was then investigated by immunofluorescence and Bradford assay. 2% chitosan discs were then enriched with increasing doses of aptamers and used as culture substrates for MC3T3-E1 cells. Cell growth was monitored by optical microscopy, while cell viability and metabolic activity were assessed by chemiluminescence and by Resazurin Sodium Salt assay. Cell morphology was investigated by cytofluorescence and by scanning electron microscopy. Chitosan films efficiently bound and retained aptamers. Aptamers did not affect the amount of adsorbed fibronectin, but affected osteoblasts behavior. Cell growth was proportional to the amount of aptamer used for the functionalization, as well as aptamers influenced cell morphology and their adhesion to the substrate. Our results demonstrate that the enrichment of chitosan films with aptamers could selectively improve osteoblasts behavior. Furthermore, our results support further investigation of this type of functionalization as a suitable modification to ameliorate the biocompatibility of biomaterial for hard tissue engineering applications.Graphical abstract

Stanozolol-soaked grafts enhance new bone formation in rat calvarial critical-size defects.

Androgen hormones play a significant role in regulating bone morphogenesis and in maintaining bone homeostasis throughout life. This study aimed to investigate the local effects of the non-aromatizable androgen stanozolol (ST) on bone regeneration in rats. Bilateral critical-size defects were created in the parietal bone of 26 male Wistar rats: the defect on one side was filled with a deproteinized bovine bone scaffold (DBB) soaked in ST solution (test) and the contralateral with DBB alone (control). Samples were collected at one month and three months. Histomorphometry revealed a significantly higher new bone formation (NB) (24.41% ± 4.14% versus 15.01% ± 2.43%, p < 0.05) and mineral apposition rate (MAR) (9.20 μm/day ± 0.37 versus 6.50 μm/day ± 1.09, p < 0.05) in the test versus control group at one month. Accordingly, real time-polymerase chain reaction revealed a consistently higher Runx2 expression in test samples (fold change test/control: 4.50 ± 1.17, p ≤ 0.05). No morphometrical differences between groups were detected at three months (p > 0.05). However, test samples were characterized by an increase in blood capillary density from one month (11.43 n mm-2 ± 2.01) to three months (28.26 n mm-2 ± 5.62), providing evidence of a vital remodeling tissue. Control samples presented a decrease of anti-Osterix (SP7)/anti-osteocalcin (BGLAP) (3.9 n mm-2 ± 0.32 versus 1.01 n mm-2 ± 0.20) and alkaline phosphatase (ALP) (12.14 n mm-2 ± 6.29 versus 6.29 n mm-2 ± 2.73) immunohistochemical-positive elements, which was suggestive of a stabilized healing phase. Based on these observations, local ST administration boosted bone regeneration in rat calvarial critical-size defects at one month. This study showed the potential of local steroid delivery in bone regeneration.

Role of prostaglandin E2 in the modulation of Wnt canonical signaling in cells on microstructured titanium surfaces

Background Rough surface topography enhances the activation of Wnt canonical signaling, a pathway required for osteoblast differentiation. The present study investigated the effects of the modulation of prostaglandin E2 (PGE2) signaling on osteoblastic differentiation on titanium surfaces for endosseous implants with different topographies. Methods C2C12 cells were plated on polished or acid-etched/sand-blasted (SLA) titanium discs and stimulated with 1 μM PGE2 or 100 nM cyclooxygenase inhibitor indomethacin. Activation of Wnt canonical signaling was measured with a reporter system. Gene expression was measured in the same cell system by real-time polymerase chain reaction (RT-PCR). Osteoblastic MC3T3 cells were then plated on polished or SLA titanium discs with or without indomethacin, and their proliferation and the expression of osteoblast-specific genes was assessed by RT-PCR. Cell morphology was furthermore studied on SEM, and cell adhesion was assessed by fluorescent labeling of focal adhesion. Results PGE2 decreased Wnt signaling stimulation in cells growing on polished or SLA surfaces, while indomethacin increased the expression of Wnt target genes in C2C12 and MC3T3 cells, by reporter assay. Moreover, indomethacin increased the expression of early differentiation marker alkaline phosphatase in MC3T3 cells on polished discs and of late marker osteocalcin in cells on SLA titanium. Conclusions Prostaglandin signaling affects the activation of Wnt canonical pathway in osteoblastic and mesenchymal cells on microstructured surfaces.