Objective. To evaluate horizontal bone gain and implant survival and complication rates in patients treated with titanium meshes placed simultaneously with dental implants and fixed over them. Methods. Twenty-five patients treated with 40 implants and simultaneous guided bone regeneration with titanium meshes  were selected for inclusion in the present retrospective multicenter study. Primary outcomes were horizontal bone gain and implant survival; secondary outcomes were biological and prosthetic complications. Results. After the removal of titanium meshes, the CBCT evaluation revealed a mean horizontal bone gain of 3.67 mm (±0.89). The most frequent complications were mild postoperative edema (12/25 patients: 48%) and discomfort after surgery (10/25 patients: 40%); these complications were resolved within one week. Titanium mesh exposure occurred in 6 patients (6/25 : 24%): one of these suffered partial loss of the graft and another experienced complete graft loss and implant failure. An implant survival rate of 97.5% (implant-based) and a peri-implant marginal bone loss of 0.43 mm (±0.15) were recorded after 1 year. Conclusions. The horizontal ridge reconstruction with titanium meshes placed simultaneously with dental implants achieved predictable satisfactory results. Prospective randomized controlled trials on a larger sample of patients are required to validate these positive outcomes. Dental implants are a predictable treatment procedure for the prosthetic rehabilitation of partially and fully edentulous patients –3.An adequate bone volume is required for insertion of dental implants , 5; the Since frequently patients present with bone defects of variable entity , 5, diffGBR is considered one of the most predictable of these techniques in terms of clinical outcomes, as reported by several systematic reviews of the literature –15, partBone regeneration with GBR has been demonstrated to be predictable, whether or not biomaterials are positioned below the membrane and are contained by it , 14, 16.An ideal membrane should possess the following characteristics: biocompatibility, space maintenance capabilities, and ease of use , 17, 18.In particular, the titanium meshes represent a valid solution, because they meet most of the ideal requirements that a membrane should possess , 15. SevThe proper placement and stabilization of the titanium mesh into the defect site is of fundamental importance for the success of the regenerative therapy , 16–18; Recently, titanium meshes that can be fixed directly on the implant have been introduced, but there is still a lack of clinical studies evaluating the efficiency and predictability of these membranes , 26.Therefore, the purpose of the present retrospective, multicenter clinical study is to evaluate the horizontal bone gain, the percentage of implant survival, and the degree of complications in patients treated with titanium meshes positioned simultaneously with dental implants and fixed over them.Patients enrolled in the present retrospective multicenter study were identified through the customized records of five different private dental clinics. Only records of patients with partial edentulism of the maxilla and/or mandible (with a period of edentulism of at least 4 months) or in need for replacement of nonrestorable failing teeth at the time of recruitment, who had been treated with titanium mesh and simultaneous implant placement in a period between January 2013 and December 2014, were reviewed. Further inclusion criteria for the present study were insufficient width of a portion of the alveolar process, with the need for horizontal augmentation of at least 3-4 mm, age > 18 years, good systemic and oral health, dentition in the opposing jaw, detailed information about the treatment, and a minimum follow-up of 1 year. In fact, the customized records of patients had to include all patient-related  and implant-related  information; in addition, they had to contain information about the occurrence of implant failures and/or biological and prosthetic complications during the entire follow-up period, since any complication that was manifested clinically was routinely referred back to the specialist practice for control. Exclusion criteria were any systemic disease that could contraindicate surgery , poor oral hygiene, and active periodontal infections. All patients had been informed about the planned treatment and had signed an informed consent form. All data were inserted into spreadsheet software and used for statistical evaluation. The study was performed in accordance with the principles outlined in the Helsinki Declaration on Human Experimentation, as revised in 2008.A preliminary clinical and radiographic examination had been performed prior to commencing the surgical procedures. All patients received a session of professional oral hygiene, with scaling and root planning, two weeks before surgery. In addition, patients were instructed about common oral hygiene procedures and were prescribed with chlorhexidine 0.2% mouthrinses, twice a day for 2 weeks, so that, before entering the surgical procedures, they all had an adequate plaque control. At the same time, a thorough radiographic examination was performed, in order to precisely assess the width of the  alveolar process. Cone beam computed tomography (CBCT) scans were taken; then, raw CBCT data were imported into reconstruction software, where a careful three-dimensional (3D) evaluation of the alveolar process was performed. Linear and volumetric measurements were obtained, in order to fully disclose the anatomy of the bone site and therefore to choose the most appropriate implant and titanium mesh for reconstruction.All patients were installed with tapered implants  characterised by strong self-cutting threads. These implants featured a 5 mm deep conical connection (10°) combined with an internal hexagon –30. The  in situ; then, they were fixed with a cover screw. An absorbable collagen membrane  could be adapted over the titanium meshes, according to the clinicians' preferences. The soft tissues were adapted over the membranes and care was taken in order to avoid tension during sutures. A tension-free closure was obtained through horizontal mattress sutures; single-loop sutures were made to further seal the incision line. Ice-packs were provided postoperatively, with the recommendation to keep them onto the treated area for at least 2 hours. Patients were prescribed oral antibiotics, amoxicillin plus clavulanic acid 1 gr every 12 hours, for 6 days. Postoperative pain was controlled by administering 600 mg ibuprofen every 12 h for 2 days. Patients were instructed to rinse with chlorhexidine digluconate 0.2%, 2-3 times per day, for an overall period of 2-3 weeks, with the recommendation to discontinue tooth brushing in the surgical area. A soft diet was recommended in this period, in order to avoid any trauma in the site of surgery; coherently, patients were asked not to wear removable dentures, where present, for a period of 1 month after surgery. Patients were recalled and checked at 2, 5, and 10 days after operation, to monitor their healing; 14 days after surgery, sutures were removed. After 3-4 months, a second-stage surgery was performed at the recipient sites. The fixtures were uncovered, and the titanium screws and meshes were removed  aspect of the ridge ; sutures Figures . Before The horizontal dimensions of the alveolar ridge were measured in the CBCT sections, before and 4 months after the surgery, in mm. Basically, before implant placement, one first linear measure was taken at the future implant location; this measure was taken where CBCT evaluation revealed the maximum bone deficiency. After the second-stage surgery for the removal of the titanium mesh, the same measure was repeated at the same location. This second measure was registered; then the horizontal bone gain was determined by the difference between the second and the first measurement.One year after implant placement, the prosthetic restorations were removed and the stability of all fixtures was verified. An implant was classified as “surviving” if still in function, without any problem, at the 1-year follow-up control. Conversely, failure to osseointegrate with implant mobility, progressive marginal bone loss due to bacterial tissue invasion (peri-implantitis), severe marginal bone loss in the absence of symptoms/signs of infection, and implant body fracture were the conditions in which implant removal was required.Early complications were those that occurred immediately after surgery, or in the immediate aftermath (1-2 weeks), such as pain/discomfort, swelling/edema, and extraoral contusion.All complications occurring from the third week after surgery, until the end of the study, were classified as late biological complications. These complications included titanium mesh exposure, partial or complete loss of the graft, and any disturbance in the function of the implant characterized by a biological process affecting the supporting tissues (peri-implant mucositis and peri-implantitis) and any peri-implant bone loss exceeding 1.5 mm, but in the absence of clinical signs of infection.Peri-implant mucositis is the condition in which soft tissue inflammation, pain, and swelling are present, but in the absence of peri-implant bone loss; conversely, peri-implantitis is the condition in which pain, suppuration, exudation, and fistula formation are present, with concomitant probing pocket depth ≥6 mm and peri-implant marginal bone loss >2.5 mm.The peri-implant marginal bone loss was calculated as previously reported –30. In bAll prosthetic complications that had affected the implant-supported restorations, from the placement of the provisional restorations and until the end of the study, were carefully registered. Mechanical complications included all complications occurring at prefabricated components (such as abutment screw loosening and abutment fracture) whereas technical complications included all complications of the laboratory-fabricated suprastructure or its materials .Patient demographics and distribution of implants were analyzed using descriptive statistics.Means and standard deviations as well as ranges and confidence intervals (95%) were calculated for quantitative variables, such as patient age, gain in horizontal dimensions of the alveolar ridge, and peri-implant marginal bone loss. Absolute and relative frequency distributions were calculated for qualitative variables, both patient-related  and implant-related . The Chi-square test was used to evaluate the differences among the groups. The level of significance was set at 0.05. The incidence of biological complications  and prosthetic complications  as well as the implant survival rate were calculated, 1 year after implant placement. The implant survival rate was calculated both at the patient and at the implant level. All computations were carried out with dedicated statistical analysis software.p = 0.3173), age (p = 0.4677), or smoking habit (p = 0.0719); however, most of the patients had no history of periodontal disease (p = 0.0278). Forty implants were placed . Thirty-one implants were placed in healed sites, while 9 implants were installed in fresh extraction sockets. The distribution of the implants is shown in p = 0.0001), were premolars (p = 0.0074), and were placed in healed ridges (p = 0.0005); the most frequently used implants were 10.0–11.5 mm in length (p = 0.0203) and the most frequent prosthetic restorations were SCs and short-span 2-unit FPPs (p = 0.0225). No differences were found in the distribution of the implants by diameter (0.0655). Forty titanium meshes were placed: 12 type A membranes , 22 type B membranes , and 6 type C membranes . An absorbable collagen membrane was employed to protect the titanium meshes in 12 cases (12/25: 48%).In total, 25 patients  who had been treated with implant placement with simultaneous GBR with titanium meshes, were selected for the present retrospective, multicenter clinical study. The distribution of the patients is illustrated in At the second-stage surgery and after the removal of the titanium meshes, the CBCT evaluation revealed a mean horizontal bone gain or augmentation of 3.67 mm . With regard to early biological complications, 10 patients (10/25: 40%) reported mild pain for the 3-4 days following surgery; however this discomfort was well tolerated with analgesics; conversely, 15 patients (15/25: 60%) had no discomfort or pain at all. Twelve patients (12/25: 48%) experienced mild postoperative edema; in 2 patients (2/25: 8%) this edema coexisted with extraoral contusion in the region. Eleven patients (11/25: 44%) had no edema at all. The mean time between implant placement and second-stage surgery  was 3.8 months. In most of the patients (19/25: 76%), healing proceeded without any delayed complication, and grafts appeared well incorporated into native bone. However, titanium mesh exposure occurred in 6 patients (6/25: 24%). In all these cases, weekly examinations were carried out, and mesh exposure was treated with a gentle cleaning of the area with an extra soft toothbrush soaked in chlorhexidine 1% gel. In addition, patients were asked to apply 1% chlorhexidine gel, 2 times per day, and were instructed to rinse with 0.12% chlorhexidine, 2-3 times per day. After this treatment, in 4 of these exposures, spontaneous coverage of the titanium membrane was found, with complete reepithelization of the areas and soft tissue closure, in a period between 3 and 4 weeks. These exposures did not prevent proper graft incorporation into native bone. In the remaining 2 cases, however, the titanium mesh had to be removed, because of nontreatable soft tissue defects followed by infection and loss of the graft. In one patient, the loss of the graft was partial, and it did not affect the survival of the implant; in the other one, however, the infection caused the complete loss of the graft and the implant. This implant failure was classified as “early failure,” because it occurred 2 months after surgery (before the connection of the prosthetic abutment) in a 45-year-old smoking female patient, without history of chronic periodontal disease. No other implant failures were reported. Among the restorations, 17 were SCs (17 implants), 6 were 2-unit FPPs (12 implants), 3 were 3-unit FPPs (6 implants), and 2 were 4-unit FPPs (4 implants), representing a total of 27 fixed partial prosthetic units available for analysis. No prosthetic complications were registered. All the 39 surviving implants were followed up for 1 year, for an overall survival rate of 97.5% (implant-based) and 96.0% (patient-based). The peri-implant marginal bone levels at the 1-year examination are reported in Several clinical studies –25 and sHowever, only a few of these studies –27 reporVon Arx and Kurt  have repIn another study of Jung and colleagues , ten patIn the study of Konstantinidis and colleagues , peri-imIn our present study, the alveolar ridge reconstruction with titanium meshes and simultaneous implant placement has proved to be a reliable and effective treatment, with an average horizontal bone gain of 3.67 mm (±0.89).This is in accordance with the contemporary scientific literature , 25–27, As reported in different systematic reviews , 15, 18,The titanium meshes used in the present study meet almost all these requirements: in fact, they are biocompatible, they are efficiently integrated with the tissue, and they can effectively prevent the colonization of the site by connective tissue. In addition, they have excellent space maintenance capabilities and they are easy to use. A membrane, in fact, should be sufficiently stiff to be able to counteract the pressure exerted by external forces , but at the same time quite malleable/easy to be adapted to the defect site , 15, 18.From the analysis of the current literature, the biological complications emerge as the main problem occurring with titanium membranes, both in the immediate postoperative and in the following months , 18–27. In this retrospective work on 25 patients, the most frequent complication, which occurred in 12 patients (12/25: 48%), was represented by the postoperative edema; the second complication was represented by postoperative pain or discomfort, which occurred in 10 patients (10/25: 40%). Both of these complications were classified as early complications; however, they were minor in nature as they could be easily managed with anti-inflammatory drugs, resolving already during the first week. The third complication per incidence (6/25: 24%) was instead represented by the exposure of the titanium mesh. The exposure of the titanium mesh is certainly one of the most insidious complications to handle, as reported in the literature , 15, 18;Our present study has limits. First, although it is based on data collected from different centers , it is retrospective: retrospective studies are not the best solution to investigate clinical issues and certainly have a lower value than prospective studies. For this reason, further prospective clinical studies or even better, randomized controlled trials will be needed to confirm our present positive outcomes. Second, our present work is based on a limited number of patients (and implants), and the implants here were followed up for a short time (1 year). Therefore, further long-term studies on a larger sample of patients will be needed to evaluate the efficacy of the present treatment and the reliability of these new titanium meshes for bone regeneration of small- and medium-sized peri-implant bone defects.In the present retrospective multicenter study, the authors have reported on guided bone regeneration with titanium meshes and simultaneous implant placement. In particular, a new type of titanium mesh that can be fixed directly on the fixture has been used for bone regeneration of small- and medium-sized peri-implant bone defects. Overall, the horizontal ridge reconstruction with titanium meshes positioned simultaneously with dental implants achieved predictable satisfactory results. In fact, after the removal of the titanium meshes, the CBCT evaluation revealed a mean horizontal bone augmentation of 3.67 mm (±0.89). Mild postoperative edema (48%) and pain/discomfort (40%) after surgery were the most frequent biological complications encountered, but these early complications were completely resolved within one week after surgery. Titanium mesh exposure occurred in 6 patients (24%): one of these patients suffered partial loss of the graft and another complete graft loss and implant failure. After 1 year from implant placement, an overall satisfactory implant survival rate of 97.5% (implant-based) and a limited mean peri-implant marginal bone loss of 0.43 mm (±0.15) were found. The present positive outcomes can be considered encouraging but must be confirmed by further long-term controlled studies on a larger sample of patients.


Aim: The aims of the present study were to determine the antibiotic susceptibility profils with particular emphasis on susceptible or resistant strains to macrolides and lincosamids antibiotics and to determine possible antibiotic resistance mechanisms occurring in group B streptococci (GBS) strains using PCR assay and disk diffusion method.Methods: A total of 62 clinical GBS strains were investigated. Antibacterial susceptibility testing was performed using the disk diffusion method and inducible resistance test for clindamycin by standard double disk diffusion or D-zone test for all isolates to differentiate macrolide resistance phenotype (M), constitutive macrolide-lincosamide-streptogramin B phenotype (cMLSB) and induced macrolide-lincosamide-streptogramin B phenotype (iMLSB). In addition, minimum inhibitory concentrations (MIC) of penicillin were determined for all isolates. Finally, possible existence of antibiotic resistance genes for erythromycin  and for clindamycin (linB) were examined among isolates using PCR assay.Results: All 62 isolates were susceptible to penicillin, ampicillin, linezolid, cefazoline and vancomycin. However, 93.5% (n=58) of isolates showed an increased MIC to penicillin. The overall rate of erythromycin resistance was 35.5% (n=22). All erythromycin-resistant isolates displayed the M phenotype . All three erythromycin resistance genes  were found in erythromycin-resistant isolates.Conclusion: It was concluded that prescribing antibiotic without antibacterial susceptibility tests should be prevented because of the high prevalence of erythromycin-resistant GBS strains and the fact that erythromycin-resistant GBS strains has shown an increased MIC to penicillin, as the drug of choice for treating GBS infections. More than 30 different erm genes have been found in various bacterial species. In Streptococcus agalactiae, erm (A) (subclass of erm (TR)) and erm (B) genes have been identified responsible for high-level resistance to erythromycin and clindamycin. In fact, the Erm enzyme, which induces cross-resistance to macrolides, lincosamides, and streptogramin B (MLSB), is known as the MLSB phenotype, which classifies into two categories, the first one is the induced MLSB resistance phenotype (iMLSB), and the second is the permanent MLSB resistance phenotype (cMLSB) . Peines too . linB isermB, ermTR, and ermA/E on transposons, these genes can travel from an organism to another [Because of the presence of antibiotic-resistant genes such as  another . AccordiIn fact, the present study was conducted to gain some knowledge about the susceptibility pattern of the GBS isolates in the area of the study. The study was performed from June 2013 to February 2014. A total of 62 clinical GBS isolates was collected, including 15 vaginal samples selected from 203 samples taken from pregnant women, one positive sample from a none pregnant women, 45 positive urine culture samples, and one positive blood culture sample. Moreover, 55 samples were collected from females and other six samples were obtained from males . After conventional phenotypic identification tests, all these samples were inoculated in Brain Heart Infusion (BHI) broth medium containing 5% blood and 18% glycerol at the temperature of –70°C. GBS identification criteria using diagnostic phenotypic tests were as follows; beta-hemolytic colonies, gram-positive, chain-forming coccus, catalase positive, a positive result for the sodium hippurate hydrolysis test, a negative result for bile esculin agar test, antibiotic resistance to bacitracin disk and SXT, and a positive CAMP reaction. DNA was extracted by thermal lysis   and a negative control sample .In order to confirm the extracted isolates that were detected by phenotypic methods, PCR assay with an internal positive control of The antibiotic susceptibility test for all samples was conducted using the following antibiotics; penicillin (10 units), ampicillin (10 µg), vancomycin (30 µg), tetracycline (30 µg), erythromycin (15 µg), azithromycin (15 µg), clindamycin (2 µg), cefazolin (30 µg), quinopriston/dalfopristin (synercid) (15 µg), chloramphenicol (30 µg), linezolid (30 µg), levofloxacin (5 µg) and doxycycline (30 µg). All antibiotics were manufactured by MAST Group Ltd. Concurrent with this test, the inducible resistance to clindamycin for all GBS isolates was determined using standardized double-disk (DD) diffusion test or D-zone test. In regard with D-zone test, the appearance of a D-shape halo on the medium was considered indicative of the inducible resistance to clindamycin, and accordingly, it was deduced whether there was a resistance to clindamycin or not. In the absence of such growth zones, the size of the non-growth zone for each antibiotic was measured and recorded. The interpretation of the susceptibility, resistance, or moderate resistance to antibiotics for each disk was performed based on the guidelines disseminated by CLSI 2014 . B resistance. Furthermore, coincidence of resistance to erythromycin and lack of resistance to clindamycin is indicative of M phenotype. Finally, the coincidence of resistance to clindamycin and moderate resistance to erythromycin is indicative of L phenotype [In the cases of clindamycin and erythromycin, the regions lower than 15 mm around both disks is indicative of cMLShenotype .The lowest concentration of antibiotic penicillin able to inhibit the isolates was determined using gridlines recommended by CLSI 2014 .ermB, ermTR, and mefA/E primers (by targeting genes resistant to erythromycin) for multiplex PCR and linB primer was used separately with the primer sequences presented in Table 1 In order to determine the specific resistance mechanisms, the PCR tests were conducted for all samples. For this purpose, The independent t-test was employed to compare the erythromycin antibiotic susceptibility and antibiotic susceptibility pattern of other antibiotics. The level of significance of the test was set at 0.05. All statistical analysis was performed using SPSS software package version 20.The results of PCR with specific primers demonstrated that all 62 isolates were GBS strains. The results of the antibiotic susceptibility test using the disk diffusion method for erythromycin, clindamycin, and quinupriston/dalfopristin alongside these results for other antibiotics and the results of the broth microdilution method (MIC) for penicillin antibiotic are presented in Table 2 B, cMLSB, and L phenotype in any strain. Furthermore, the results of multiplex PCR assays revealed that ermTR, ermB, and mef A/E genes were presented in 16.1, 19.4, and 12.9% of strains, respectively. The gene inducing resistance to clindamycin, linB, was identified in none of the strains. Simultaneous existence of genes resistant to erythromycin in GBS isolates are presented in Table 3 The disk diffusion method was applied in order to determine the susceptibility pattern of erythromycin and clindamycin antibiotics; finally it was found that 22 strains (35.5%) were phenotype M, and also, there were not found iMLSIn the present study, all 62 clinical isolates that were examined using the disk diffusion method were susceptible to the antibiotics penicillin, ampicillin, linezolid, cefazolin and vancomycin. However, most of them, 58 cases (93.5%), had the increased minimum inhibitory concentration for penicillin. ermB, ermTR, and mefA/E, were found in all isolates, which were resistant to this type of antibiotic. The presence of these genes was significantly related to the erythromycin susceptibility pattern (p<0.05) , 12 (19.4%), and 8 (12.9%) cases, respectively. There were not observed any linB gene, which induce the resistance to clindamycin antibiotics. The presence of these three genes was not identified in isolates. In contrast, the simultaneous presence of the ermTR and the ermB genes was identified in three isolates, while only one isolate contained both ermB and the mefA/E genes simultaneously. Furthermore, five isolates (8.1%) had the non-growth zone less than 17 mm (defined as the susceptibility limit criteria) for vancomycin disk. The overall frequency of resistance to erythromycin was 35.5% (22 cases). All isolates resistant to erythromycin were displayed M phenotype. All three genes inducing the resistance to erythromycin antibiotics  Table 3 . HoweverB phenotype, 8 cases (42%) were iMLSB phenotype, and one case was M phenotype. The ermmethylase genes are related to the presence of mefA gene and were only found in M and MLSB phenotype [The resistance to azithromycin, chloramphenicol, and levofloxacin was observed in 26, 2, and 3 cases, respectively, also all isolates were resistant to doxycycline, and tetracycline. It is interesting to note that none of the isolates were resistant to antibiotics clindamycin and quinupristin/dalfopristin. In this regard, current concerns are the resistance to macrolides and lincozamides that have been globally increased among GBS strains over two past decades; the frequency of resistance to erythromycin and clindamycin used to be less than 5%, but in recent years there have been reports explaining that these frequencies have been increased up to 54% in the United States. In Belgium, the prevalence of resistance to macrolide antibiotics among the invasive isolates increased from 10.4% in 2000 to 33% in 2008 . These fhenotype .ermTR and ermB genes were identified in 13 and 16% of isolates, but there were no ermA and mefA in the isolates. All isolates resistant to erythromycin were cMLSB phenotype, 22 genes contained only the ermTR gene and 5 isolates contained both ermB and ermTR genes [The results of other similar studies carried out in Iran are as follows; 115 isolates were analyzed by Emaneini et al., they reported that all the cases were susceptible to penicillin and quinupriston/dalfopristin antibiotics, but the level of their susceptibility to clindamycin, chloramphenicol, erythromycin, linezolid, and tetracycline were 35, 45, 35, 1, and 96%, respectively. Besides, in that study, the The difference between the results gained by that study and those obtained in the present study can be attributed to the geographical differences between the regions the studies have been done. According to the CLSI guidelines, the presence of the beta-hemolytic Streptococcus isolates resistant to betalactam antibiotics have not been identified yet . HowevermefA/E gene was the only contributor in developing M phenotype, while these results are compatible with the results obtained by Seo et al. who reported that mefA, mefB, and ermA were associated with M phenotype [In this study, the results of MIC examinations indicated that these 58 isolates had the MIC non-susceptible to penicillin . The M phenotype . Furthermore, in the present study, there was not found any significant association between the erythromycin antibiotic susceptibility pattern and the susceptibility pattern of other antibiotics other than azithromycin, because of the similarity of families, (p>0.05). Two isolates were concurrently resistant to chloramphenicol and erythromycin, but a significant association between resistance patterns of these two antibiotics was not observed. Likewise, two isolates of three ones that were resistant to levofloxacin were concurrently resistant to erythromycin, but there was no significant association between them (p=0.067) Table 6 .In this study, it was found that a high proportion of the isolates had an increased MIC to penicillin. The incidence of GBS isolates with a reduced MIC to penicillin suggests that there is a possibility of future failure in treating the infections caused by these bacteria, especially in the case of consuming these antibiotics before or during delivery. According to the results obtained in this study, the antibiotics clindamycin, quinupriston/dalfopristin, linezolid, levofloxacin, vancomycin, and, chloramphenicol can be reliable alternatives of erythromycin in treating or preventing GBS infection in women who suffer from colonization or patients that are allergic to penicillin in Hamadan.The authors declare that they have no competing interests.


In addition, the performances of the proposed MEMS electrochemical seismometers with feedback were compared to a commercial electrochemical seismic sensor (CME 6011), producing higher bandwidth (0.005–20 Hz vs. 0.016–30 Hz) and lower self-noise levels ) in the low-frequency domain. Thus, the proposed device may function as an enabling electrochemical seismometer in the fields requesting seismic monitoring at the ultra-low frequency domain.Electrochemical seismic sensors are key components in monitoring ground vibration, which are featured with high performances in the low-frequency domain. However, conventional electrochemical seismic sensors suffer from low repeatability due to limitations in fabrication and limited bandwidth. This paper presents a micro-fabricated electrochemical seismic sensor with a force-balanced negative feedback system, mainly composed of a sensing unit including porous sensing micro electrodes immersed in an electrolyte solution and a feedback unit including a feedback circuit and a feedback magnet. In this study, devices were designed, fabricated, and characterized, producing comparable performances among individual devices. In addition, bandwidths and total harmonic distortions of the proposed devices with and without a negative feedback system were quantified and compared as 0.005–20 (feedback) Hz vs. 0.3–7 Hz (without feedback), 4.34 ± 0.38% (without feedback) vs. 1.81 ± 0.31% (feedback)@1 Hz@1 mm/s and 3.21 ± 0.25% (without feedback) vs. 1.13 ± 0.19% (feedback)@5 Hz@1 mm/s (n A seismic sensor functions as a velocity sensor or an accelerometer that senses the ground vibration of the earth, which is widely used in the field of earthquake monitoring, resource exploration, and ocean bottom observation ,2,3. TheThe first prototype of the electrochemical seismic sensor was based on “solion,” which was initially developed in the 1950s by the US Navy, targeting the detection of low-frequency acoustic waves ,21,22. STo resolve these issues, the electrochemical seismic sensors based on MEMS technologies were introduced by He et al. ,26,27, wIn this paper, a force-balanced negative feedback system was proposed to extend the working bandwidth of the MEMS-based electrochemical seismometers, which, at the same time, retains the characteristics of high repeatability. In addition, the microfabrication of the sensing electrodes was improved to enhance production yield. The structure of this paper is organized as follows. In I2) and potassium iodide (KI), flows through the via-holes in the sensing electrodes. The following reversible electrochemical reactions occur on the surfaces of the anodes and the cathodes, respectively, when a DC bias (0.1–0.3 V) is applied on the electrodes, generating ion concentration gradients between each pair of the anode and the cathode.I2 with In response to external seismic signals, the electrolyte solution moves opposite to the direction of the external vibration due to the inertial force, which causes the variation of the ion concentration gradients between the anode–cathode pairs. These concentration gradients lead to current outputs, which are proportional to the external seismic signals .SW, ACW, and PFW represent the transfer functions of the sensor, the amplifying circuit, and the pre-filter, respectively), and The feedback unit composes of a feedback circuit , a feedback magnet and a feedback coil see . The rawIn a deep negative feedback (As shown in Equation (4), the output signals of the seismic sensor are shaped by The pre-filter and the PID adjuster (proportion integration differentiation) in the feedback circuit provides compensations to enhance both the amplitude and the phase margins by adjusting the values of the compensation capacitors. The increases of both amplitude and phase margins enable the system to function in a manner of deep feedback. Additionally, since the feedback force counterbalances the movement of the inertial mass in response to environmental vibrations, the dynamic range of the developed electrochemical seismometer is effectively enlarged.I2 to KI was between 1:50 and 1:100, which ensures that I2 molecules were fully dissolved in the KI solution in the forms of The sensing electrodes were fabricated by conventional MEMS technologies including lithography, deep reactive ion etching, thermal oxidation, sputtering, and wire bonding, where detailed processes can be found in previous publications . Then, tThe characterizations of the MEMS electrochemical seismic sensors were conducted on a modified home-developed platform see , which cdevice = 6). It was speculated that at a frequency higher than 1 Hz, the noise of the seismic sensor mainly comes from mechanical and environmental noises due to packaging. Thus, the increases in the self-noise levels of the proposed devices may result from imperfect packaging technologies in comparison to commercial seismic sensors.The tests of the self-noise levels were also conducted in this platform without inducing vibrations actively. As shown in A MEMS-based electrochemical seismic sensor with ultra-broad frequency working bandwidth was demonstrated in this paper. The working bandwidth and the self-noise level of the developed device were characterized with feedback parameters optimized. Experimental results showed that the working bandwidth of the developed seismic sensor was extended to 0.005–20 Hz, validating the positive effects of the negative feedback. In addition, the developed device produced a lower self-noise level at the low frequency domain in comparison to the commercial seismic sensor of CME6011, validating its large dynamic ranges. Thus, this MEMS electrochemical seismic sensor can be potentially used in the field requesting seismic monitoring at the ultra-low frequency domain with large dynamic ranges and low noise levels.


The plasma levels of serotonin, ghrelin, and peptide YY (PYY) were significantly decreased in patients with PCOS compared with controls, and have a significantly negative correlation with waist circumference and testosterone. Sequencing of the V3–V4 region of the 16S rRNA gene in fecal samples revealed the substantial differences of gut microbial species between the PCOS and non-obese controls. Bacterial species were clustered into 23 co-abundance groups (CAGs) based on the SparCC correlation coefficients of their relative abundance. The CAGs increased in PCOS, including the bacteria belonging to Bacteroides, Escherichia/Shigella and Streptococcus, were negatively correlated with ghrelin, and positively correlated with testosterone and BMI. Furthermore, the CAGs that were decreased in PCOS, including the bacteria from Akkermansia and Ruminococcaceae, showed opposite relationship with body-weight, sex-hormone, and brain–gut peptides. In conclusion, gut microbial dysbiosis in women with PCOS is associated with the disease phenotypes.Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder in women. Gut microbiota has been implicated to play a critical role in metabolic diseases and may modulate the secretion of mediators of the brain–gut axis. Interaction between gut microbiota and the endocrine and biochemical disturbances in PCOS still remains elusive. Here, we showed an altered gut microbiota significantly correlated with PCOS phenotype. There were 33 patients with PCOS (non-obese PCOS individuals, PN,  Polycystic ovary syndrome is a common heterogeneous endocrine and chronic metabolic disease affecting 3–26% of reproductive-aged women by applying the different diagnostic criteria . Women win vitro and in vivo  or without PCOS (n = 15) were then stratified according to BMI into the following four subgroups: obese PCOS patients , non-obese PCOS patients , obese controls  and non-obese controls . Obesity was defined as BMI ≥ 25 kg/m2 and the non-obesity otherwise according to the World Health Organization  between January 2014 and July 2014. Diagnosis criteria for PCOS established by Chinese Medical Association was based on Rotterdam criteria and the currently available evidences in Han Chinese. In our study, to avoid the confusion due to the different diagnostic criteria, each participant had met the three features based on Rotterdam criteria and the Chinese criteria as follows: oligo- or anovulation, clinical and/or biochemical signs of hyperandrogenism, and polycystic ovaries . Women inization . The stug for 15 min at 4°C, and were transferred into separate tubes and acidified by adding 5 N HCl to a final concentration of 0.05 N. The processed serum samples were stored at -20°C until analysis. For PYY, blood samples were immediately added DPP-IV inhibitor and aprotinin to a final concentration 500 KIU/mL. Fecal samples were collected on the same day. Feces was divided into aliquots and was frozen on dry ice immediately upon collection and stored at -80°C until analysis.All subjects were examined in the morning after overnight fasting (≥8 h). 4-(2-aminoethyl)-benzenesulfonyl fluoride was immediately added to the blood samples for enzyme-linked immunosorbent assay (ELISA) of ghrelin at a final concentration of 1 mg/mL. After clotting at room temperature for 30 min, the blood samples were centrifuged at 3,000 ×Waist circumference was measured midway between the lower costal margin and the iliac crest, and hip circumference at the level of maximum extension of the buttocks. WHR was calculated as waist circumference divided by hip circumference. BMI was calculated as body weight in kilograms divided by body height squared in meters.Biochemical indexes were measured on automatic biochemical analyzer . HbA1c level was measured by high-performance liquid chromatography . FINS, FSH, LH, and testosterone were tested using an automated immunoassay system . The HOMA-IR and beta cell function (HOMA-beta) were calculated using the following formula:Plasma serotonin, ghrelin, and PYY levels were determined with the commercially available ELISA kit according to manufacturer’s instructions. The kit for serotonin was purchased from EUROIMMUNUS, Inc. , and kits for ghrelin and PYY were bought from EMD Millipore Corporation . The intra-assay and inter-assay coefficients of variation were <5 and <10, respectively.The investigation of mental psychological health was conducted with SDS and SAS.DNA extraction from each frozen fecal sample was conducted as previously described . All samt-test or a one-way analysis of variance (ANOVA) for normally distributed variables or non-parameter Wilcoxon test for non-normally distributed, with multiple comparisons performed using SNK (Student-Newman-Keuls) correction. Partial correlation analysis was used to study the relationships between brain–gut peptides and other metabolic parameters. A multiple stepwise regression analysis was employed to identify influence factors of brain–gut peptides, using a stepwise selection method with a P-value of 0.15 at entry and a P-value of 0.15 at stay. All independent variables in the multiple regression were tested for multicollinearity. Statistical tests were based on two tailed probability. A P-value less than 0.05 was considered statistically significant. Power calculation was performed using PASS software 11.Statistical analyses were performed using SAS statistical software 9.2 . Normal distribution of the data was calculated with the Kolmogorov–Smirnov test. Continuous variables were expressed as mean ± standard deviation (SD). Variables that were not normally distributed were rank transformed before analysis. Differences on clinical characteristics and metabolic biomarkers were evaluated using 1. Quality-filtered sequences were clustered into unique sequences and sorted in order of decreasing abundance to identify representative sequences, and singletons were omitted in this step. Chimera detection was performed using UCHIME  to equal the difference in sequencing depth. The rarefaction estimates, the Shannon index, the Simpson’s diversity index and the Chao1 index were calculated using QIIME v1.2.1 , and theTo pick key OTUs that might be responsible for the segregation, RDA was performed using the R ‘vegan’ package. Statistical significance was assessed by Monte Carlo test with 9999 permutations.P < 0.001). Permutational MANOVA was performed using the R ‘vegan’ package2 and the CAG network was visualized in Cytoscape. Spearman correlation between CAGs and clinical parameters was calculated using MATLAB, and Benjamini-Hochberg method was used to control false discovery rate . Plasma serotonin level was significantly negatively correlated with waist circumference, hirsutism score, testosterone and SAS value, whereas, significantly positively correlated with neutrocyte count, HOMA-beta and TCH. Plasma PYY level was significantly negatively correlated with waist circumference, WHR, hirsutism score, testosterone, PPG, ALT and AST, whereas, significantly positively correlated with neutrocyte count and HOMA-beta. Plasma ghrelin level was significantly negatively correlated with waist circumference, WHR, hirsutism score, testosterone, PPG, P2hINS, ALT, and AST. To explore whether plasma brain–gut peptide/indole levels were independently associated with the clinical parameters, multiple stepwise regression analysis involving all the parameters with significant correlations with the mediators of the brain–gut axis was performed . Adjusting for age and BMI, it revealed that the testosterone level , neutrocyte count  and waist circumference  were independently associated with plasma serotonin levels; the waist circumference , the PCOS group  and neutrocyte count  were independently associated with plasma PYY levels; while the testosterone level , the PCOS group , BMI , and P2hINS  were independently associated with plasma ghrelin levels in all participants. Thus, the levels of the brain–gut peptides and the brain–gut indole were correlated with the PCOS related phenotypes.In order to evaluate the association between the mediators of the brain–gut axis and the various metabolic characteristics of PCOS, we calculated the partial correlation between serotonin, PYY, ghrelin and the clinical parameters of all subjects after adjusting for age and BMI , while no difference was found for the Shannon index and the Simpson’s diversity index. CAP and MANOVA based on the Bray–Curtis distance revealed a separation between CN group and the other three groups . Notably, the gut microbiota composition of CO group was more similar with that of PN and PO groups, rather than CN group. CAP and MANOVA based on the UniFrac distance also showed similar trends  were obtained from all 48 samples. 770,892 high-quality reads  were clustered into 567 OTUs at 97% similarity level. According to observed OTUs and the Chao1 index, CN group owned the highest richness, followed by CO and PN group, and PO group showed the lowest richness of gut microbiota . We identified 28 key OTUs that responded to the differentiation between the four groups according to RDA . OTU4 belonging to genus Bacteroides was enriched in PO group than CN and CO groups , with PN group falling in between them. The average relative abundances of 21 OTUs were higher in CN group than PO group (P < 0.05), which mostly belonged to the genera Akkermansia, Bacteroides, Clostridium IV, Lactobacillus, Oscillibacter, or unclassified genera from family Ruminococcaceae, while in CO and PN groups, the abundances of these OTUs were lower than CN group but were higher than PO group. Five OTUs were enriched in CO group, which belonged to the genera Collinsella, Paraprevotella, Slackia, and unclassified genera from class Clostridia and kingdom Bacteria. OTU7 belonging to Bacteroides showed decreased abundance in CO group, compared with the other three groups .We then used RDA to identify the gut bacteria responding to PCOS and obesity. RDA confirmed that both PCOS and obesity were significantly correlated to the dysbiosis of gut microbiota , 3, 15, 22, and 23, respectively. According to the Kruskal–Wallis test, 14 CAGs showed no significant difference between the four groups. CAG1, 4, and 7 were enriched in PO group rather than CN group. CAG 1 contained 14 OTUs, which belonged to genera Bacteroides, Escherichia/Shigella, Streptococcus, Blautia, and Parabacteroides. CAG4 contained 5 OTUs, which mainly belonged to Clostridium XlVa, Alistipes and Weissella. CAG7 contained OTUs belonging to Granulicatella, Streptococcus, Peptostreptococcus, and Rothia. While, CAG10, 11, 12, and 13 decreased in PO group rather than CN group. The dominant OTUs in CAG10-13 mostly belonged to Akkermansia, Alistipes, Coprococcus, and Ruminococcus. In CO and PN groups, the relative abundances of all these above CAGs fell in between those in PO and CN groups. In addition, CAG17 and 18 showed increased trends in CO group compared with the other groups .As the bacteria act as interdependent functional groups (guilds) in the gut ecosystem , we consFigure 5). The results showed significant correlation between gut microbiota and PCOS related symptoms, including obesity, inflammation, hyperinsulinmia, hyperandrogenism, abnormal ghrelin level, and psychological state. CAG1, 4 and 7, which were enriched in PO group, had a positive correlation with the disease phenotypes; while CAG10–13 and 18, which were enriched in CN group, had an opposite trend.To explore the relationship between gut microbiota and host phenotype, Spearman correlation between 27 clinical parameters and 9 CAGs that showed significant difference among the four groups  produced by gut microbiota  and the observation of association but not causality, further studies are needed to enlarge sample size and explore the potential causal mechanisms between gut microbiota and PCOS, such as the metabolites produced by gut microbiota and fecal microbiota transplantation.Taken together, our work suggested an altered composition of gut microbiota, represented by the reduction of alpha diversity, the increase of LPS-producing bacteria, and the decrease of spore-forming species. This study also demonstrated the association between gut microbiota and PCOS-related clinical parameters. The previous study did not find any association of the saliva microbiota and PCOS phenotypes , indicatThis study was carried out in accordance with the recommendations of Guideline on Informed Consent, Human Research Ethics Committee of Shanghai General Hospital with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Human Research Ethics Committee of Shanghai General Hospital.The authors XD, YP, CZ, and RL conceived the study and designed the experiments. YS, JS, FZ, LL, XW, YL, HF, and WD performed the technical procedures. RL and XD provided the interpretation of data and wrote the paper. AG, AR, LZ, and XD revised the final draft of the paper. All authors read and approved the final version of the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Intellectual disability (ID), characterized by impairments in intellectual function and adaptive behavior, affects 1-3% of the population. Many studies investigated its etiology, but few are cohort studies in middle-income countries.To estimate prevalence, etiology, and factors related to ID among children prospectively followed since birth in a Southern Brazilian city (Pelotas).In 2004, maternity hospitals were visited daily and births were identified. Live-born infants  whose family lived in the urban area have been followed for several years. At the age of 2 and 4 years, performances in development and intelligence tests were evaluated using the Battelle Developmental Inventory and Wechsler Intelligence Scale, respectively. Children considered as having developmental delay were invited to attend a genetic evaluation.At 4 years of age, the prevalence of ID was 4.5%, and the etiology was classified into 5 groups: environmental (44.4%), genetic (20.5%), idiopathic (12.6%), neonatal sequelae (13.2%), other diseases (9.3%). Most children presented impairment in two or more areas of adaptive behavior. There was no difference in prenatal care attendance or maternal schooling among the groups.For about 40% of children, ID was attributed to nonbiological factors, suggesting that the rate may be reduced with appropriate interventions early in life. Intellectual disability  is characterized by a significant impairment in intellectual function, associated with impairment in adaptive behavior: communication, domestic personal care, social skills, use of community resources, autonomy, health and safety, school, leisure, as well as work . The onsThe etiologic determination of ID is important for treatment, genetic counseling, family planning and support, as well as surveillance and implementation of preventive programs. It has been estimated that ID prevalence ranges from 1 to 3% ,3, but wMost of the evidence on ID prevalence and etiology comes from high-income countries ,8. FurthIn 2004, maternity hospitals in Pelotas, Brazil, were visited daily, and the births were identified. Live-born infants whose family lived in the urban area of the city were examined and their mothers interviewed  . Face-toCohort members were followed up at 3, 12, 24, and 48 months. The 3-month assessment was partially conducted in a research clinic, and other assessments were carried out at participants' home. In each visit, the children were weighed and examined, and their mothers were interviewed. Maternal depressive symptoms were evaluated using the Edinburgh Postnatal Depression Scale (EPDS) , which wStimulation was assessed at 12, 24, and 48 months using a 5-item questionnaire that evaluated cognitive stimulation markers, parent-child interactions, and interpersonal interactions ,14. The At 12 and 24 months, child development was assessed using the screening version of the Battelle Developmental Inventory (BDI), which included five domains: personal - social; adaptive; fine and gross motor; communication, and cognitive . In the The genetic evaluation was conducted at the age of 7-8 years among children who met at least two of the following criteria:BDI <-1 standard deviation (SD) at the 24-month evaluation.One or more of the following problems noted by the interviewer at the 48-month visit: communication impairment (comprehension/language); aggressive behavior; no interaction with interviewer, and inability or refusal to complete WPPSI.Intelligence quotient (IQ) below 70 points at the 48-month visit, based on the WPPSI scale.Fifteen children who did not complete the WPPSI evaluation were also invited to attend the clinical evaluation.Multiple criteria were used to increase screening sensitivity and identify children at risk of ID. Some children met multiple criteria fig. .Among 195 children who attended the clinic visit, 44 children were excluded from the analysis because their score on the BDI was <-2 SD at 24 months of age. However, at the age of 7-8 years, these same children showed a WPPSI score >70, did not present dysmorphic features or behavioral problems, and their school performance was adequate. These children were considered as having temporary delay, probably related to low birth weight (11.4%), prematurity (25.6%), HIV (13.6%), and 5-min Apgar score <6 (5.7%).Children with suspected ID were evaluated in a child friendly environment by a pediatrician/clinical geneticist, who collected information on social and demographic family characteristics, child clinical history, and family pedigree, including at least the last three generations. During the mother's interview, the child's interaction with the family, the investigator, and the environment was observed. Furthermore, the clinical geneticist carried out a physical and a dysmorphological examination and evaluated adaptive behavior by asking questions based on the Vineland-II scale . InformaCytogenetic analysis at 400-500 G-band resolution (GTG karyotype). This analysis is for children who presented a possible ID associated to minor or major anomalies or a classic phenotype syndrome such as trisomy 21. A total of 47 tests were analyzed.Molecular cytogenetic tests. These tests, e.g. fluorescent in situ hybridization, were requested when classical microdeletion syndromes were suspected. Five tests were performed.Array-based comparative genomic hybridization (array-CGH or a-CGH). This is a whole-genome analysis using the Agilent 60-mer oligonucleotide-based microarrays with a resolution of 60 Kb. It was performed for those subjects whose GTG karyotype was normal, but with a suspicion of structural abnormalities or microdeletion/microduplication syndromes. A total of 31 tests were performed.Polymerase chain reaction. This test amplified the region containing a CGG  or GCC  repeat in fragile X syndrome. Polymerase chain reaction was requested for males (n = 18) with the following characteristics: prominent ears, elongated face, poor visual contact, hypersensibility, macroorchidism, or for males with unexplained ID and normal GTG karyotype, considering the literature recommendations and the fact that the above features could not be present at the age of 7-8 years ,3.Biochemical tests. The following tests were requested for children with suspected metabolic disorders: screening tests for inborn errors of metabolism in urine; gas chromatography for organic acids in a random morning sample; high-performance liquid chromatography of amino acids in plasma and/or urine; serum transferrin in gel isoelectric focusing, and acylcarnitine profiles (liquid chromatography associated with tandem mass spectrometry) in dried blood spots. Biochemical tests (n = 6) were performed based on findings in history and/or clinical examination as recommended ,3. OrganChildren with suspected ID were classified into one of the following groups, according to the possible etiology:Genetic: based on clinical and/or laboratory diagnosis of a genetic disorder.Idiopathic: those children with syndromal characteristics, such as dysmorphisms and abnormal behavior with normal laboratory tests and inconclusive clinical evaluation.Potential neonatal sequelae: history of anoxia based on information gathered from one of the following sources: neonatal card; perinatal questionnaire; hospital records and/or image exams; hypoglycemia; intracranial bleeding; neonatal meningitis; birth injury, or extended hospitalization during the neonatal period associated or not to prematurity.Other diseases: children who did not present any complication during delivery or in the first days of life, but were diagnosed as having epilepsy, and visual or hearing impairment.Nonbiological causes: no evidence of genetic disorders, neonatal sequelae, syndromal features, or any other disease, combined with socioaffective deprivation and a poor school performance.Children's relatives  with suspected ID were also evaluated when familial history suggested that they could be potentially affected. Congenital infections were discarded based on maternal records and children examination.The institution's Ethics Committee approved this study, and the guardians of all participants signed an informed consent form.2 test was used to compare proportions. Multinomial logistic regression was used to estimate the risk of being in a category of ID. In this analysis, the nonbiological group was used as a reference.Analysis was performed using Stata 13.0. The average IQ at 48 months was compared using analysis of variance (ANOVA), the assumption of homogeneity of variances was assessed using Levene's test. χConcerning the socioeconomic variables, parental schooling was assessed through the total of completed years of formal education, and socioeconomic level was evaluated using the Brazilian Association of Market Research Institutes (ABEP) classification, in which the highest level is designated A and the lowest is E. This classification assesses the possession of household goods and the head's of the household schooling time.To estimate ID prevalence, the number of children who fulfilled the inclusion criteria (n = 170) was divided by the number of children interviewed at the age of 48 months , excluding those 44 children with temporary delay.For the present study, 214 children with suspected ID were invited to visit the research clinic to be evaluated by a pediatrician/clinical geneticist (S.M.K.). A total of 195 children (91.1%) were evaluated, with an ID prevalence of 4.5%. IQ was assessed in 3,784 children in the total of the cohort study, and the average IQ was 96.6. With respect to ID etiology, among the 195 children who attended clinical evaluation, 44 were excluded because they were considered as having a temporary developmental delay, as previously described. Information on ID etiology was obtained for 151 children. These children were classified in five groups according to ID etiology: genetic, idiopathic, potential neonatal sequelae, other diseases, and nonbiological. Most pregnancies were planned, access to prenatal care was nearly universal, more than 58% of mothers had their first antenatal care visit in the first trimester of pregnancy, and most mothers had seven or more antenatal care visits. Considering the number of previous miscarriages, the difference among groups was not statistically significant (p = 0.14).Concerning distribution of neonatal characteristics among the five groups (data not shown), birth weight, Apgar score, and gestational age were lower among children with potential neonatal sequelae (p < 0.001).Table www.karger.com/doi/10.1159/000448912).Table This study evaluated the prevalence of ID and its etiology in a population that has been prospectively followed since birth. The studied population included almost all live-born infants (99.2%) delivered in 2004, whose family lived in the urban area of Pelotas, and an ID prevalence of 4.5% was found.Most studies on ID prevalence have been carried out in high-income countries, based on clinic or administrative data, and did not evaluate the etiology of ID . SimilarGenetic disorders were found in approximately 20% of all investigated children, which is the second most frequent cause of ID, slightly below from what was mentioned in other studies . Down syMore than 40% of children with suspected ID did not have any medical condition that could explain a delayed performance in the intelligence test. Several studies have reported that family income and low maternal schooling are the strongest predictors of developmental delay . ParentaThis study has some methodological limitations. Data collected in the first 4 years of life were used to identify children with ID at the age of 7-8 years, and it is possible that additional ID cases may have emerged between the ages of 4 and 6-7. Array-CGH and exome sequencing were unavailable for all children and both tests could explain some other cases. Moreover, 8.8% of children with suspected ID were not able to be followed, but these children were equally distributed according to birth weight, gestational age, Apgar score, maternal age, and maternal schooling (data not shown).Besides the limitations discussed above, this study presents some advantages as the unlikeliness of the selection bias due to a high proportion of successfully followed cohort members . The likChildren belonging to the group of nonbiological causes have never attended any kind of intervention/stimulation program. Early interventions are designed to support young children who are at risk or who have been identified as having disabilities. In addition, the importance of these interventions, independently of ID etiology, has also been observed . In BrazThe authors declare that there are no conflicts of interest to disclose.


Resting HR increased overall significantly at D1 , due to a significant decrease in parasympathetic drive. This decrease was in FIN only partially recovered toward POST. In FIN only, baseline HR was negatively correlated with mean velocity [r −0.63 (P.04)] and parasympathetic drive [pNN50+: r −0.67 (P.03)], a lower HR and a higher vagal tone predicting a better performance. Moreover, in FIN, a persistent increase of the long-term self-similarity coefficient, assessed by detrended fluctuation analysis (DFAα2), was retrieved, possibly due to higher alertness. As for psychometrics, at D1, POMS Vigor decreased , while Fatigue augmented . Sleepiness increased only in NON, while Borg scales did not exhibit changes. Baseline comparison of mood states with normative data for athletes displayed significantly higher positive mood in our athletes. Results show that: the race conditions induced early decreases in parasympathetic drive; the extent of vagal withdrawal, associated to the timing of its recovery, is crucial for success; pre-competition lower resting HR predicts a better performance; psychological profile is reliably depicted by POMS, but not by Borg fatigue scales. Therefore, assessment of heart rate variability and psychological profile may monitor and partly predict performance in long-duration ultramarathon in extreme cold environment.Studies on human physical performance in extreme environments have effectively approached the investigation of adaptation mechanisms and their physiological limits. As scientific interest in the interplay between physiological and psychological aspects of performance is growing, we aimed to investigate cardiac autonomic control, by means of heart rate variability, and psychological correlates, in competitors of a subarctic ultramarathon, taking place over a 690 km course (temperatures between +5 and −47°C). At baseline (PRE), after 277 km (D1), 383 km (D2), and post-race , heart rate (HR) recordings , psychometric measurements  were obtained in 16 competitors . As not all participants reached the finish line, comparison of finishers (FIN,  Human physiology is characterized by continuous reactive adaptation to internal and external conditions and stressors  long-term strenuous exercise, (ii) extreme cold exposure, and (iii) impaired resting conditions, due to in-race camping. So far, among studies on ultra-endurance exercise, research objectives mostly diverge from evaluation of autonomic cardiac function in ultramarathon runners , often under strenuous environmental conditions, with concurrently impaired possibilities to recover. The Yukon Arctic Ultra (YAU) is considered to be one of the world's toughest ultramarathons  were of Caucasian descent and one was of Asian origin. Their anthropometric data are presented in Table From a total number of 78 athletes partaking in the 690 km foot-race category of the YAU during the years 2013, 2015, and 2017, 27  volunteers enrolled in the study . Due to issues related to data collection, from the 27 participants, only 16  were included in the data analysis (see section Statistics). The majority , and all measurements and procedures complied with the Declaration of Helsinki All study participants included in the final analysis had completed either one marathon (9.6 ± 24.4) or ultramarathon (14.4 ± 24) prior to their study-participation. The mean longest ultramarathon distance completed by the athletes before their YAU participation was 380 ± 220 km. In addition, seven of the study participants had previously participated in the YAU foot-race in various distance categories, with a mean longest completed distance of 478 ± 219 km. Thus, the study participants were experienced endurance athletes, which is also reflected by their self-reported sedentary HR of 52.6 ± 7.3 bpm. From one participant, this background data was not made available.The Montane® YAU ultra-endurance race takes part in the beginning of February, covering a 690 km distance between Whitehorse and Dawson City in the Canadian Yukon Territory. Besides the foot-race, the YAU also allows the competition in cross-country-skiing and mountain-biking. The first and last sections of the trail account for elevations between 500 and 700 m, however, especially in the last 200 km, the terrain along the Yukon river partly exhibits great elevation gains up to 1,000 m). The YAU is not an orientation race, as the trail is marked and prepared with snow-mobiles. Via GPS devices, athletes can be tracked on the course and have the possibility to call for assistance in case of an emergency. To further increase their safety, the time until the race has to be completed is limited to 14 days and additionally, medical screenings are administered at the 10 checkpoints which are located (mostly about 50 km apart) along the route. Despite these partly indoor vacancies , during the race, competitors face complete exposition to the subarctic environment, with outdoor temperatures in February ranging between +5°C (highest temperature measured in February 2013 in Whitehorse) and −47°C (lowest temperature measured in February 2015 in Dawson City). Additionally, the extremely high air humidity (up to 100% as measured in February 2013 in Whitehorse) contributes to the possible onset of frostbite, which, along with other  concerns, may lead to immediate disqualification. Importantly, the weather conditions between editions were not significantly different  and, apart from the checkpoints, had to eat, rest and make toiletry arrangements in the outdoor conditions of the Yukon Territory.More detailed information about the Montane® YAU is provided on the official website of the eventExperimental protocol details are depicted in Figure The in-race checkpoints had to be selected for measurement implementation due to essential practical concerns. They had to be indoors facilities buildings with sufficient space, comfortable ambient temperature and low noise in order to perform measurements under controlled conditions, as well available electricity and that it was accessible by car for the investigators. Exemplarily, several of the race checkpoints were mere tents that did not meet these criteria and therefore, the study checkpoints were chosen as they were. Thus, the distance between the race start  and the first in-race assessment D1) accounted for over a third (277 km) of the entire race-course and additionally, in this period, athletes would face the most strenuous weather conditions , baseline anthropometric data  were obtained. Weight was measured using a calibrated scale  and height was taken from the participants' interview.Fifteen minutes baseline recordings of beat-to-beat HR to assess HRV were collected with a HR monitor , which is widely used and validated for HRV assessment  and upon awakening , HR data was collected, as at baseline, to assess HRV. Afterwards, before departure, psychometric assessment again took place  monitored by means of a heart rate monitor .time (h)/space (km) = velocity (km/h)]. By collection of in- and out-going times at in-race measurement points for each participant, both split times and velocities could be computed, allowing detailed assessment of performance. Additionally, continuous HR measurements served to determine exercise intensity, as well as resting quality . The continuous HR recordings, collected during the race, were screened for quality . The average, maximum and minimum HR were determined per each selected race period, and the values were normalized with respect to the individual age-related maximal HR (HRmax)  and average resting HR (RestHR). This approach was selected to allow comparison with parameters assessed at checkpoints  and, by classifying data, served to better interpret findings.The official time at the end of the race for each participant who reached the finish line was collected, together with the times and the respectively completed distance for each participant who had to withdraw. Subsequently, the mean running velocity of the race was calculated from the total recorded time and the total distance covered /SD. This transformation converted raw scores to normalized scores on a standard scale with a mean of 50 and a standard deviation of 10, so that individual results could be compared with normative sample data. POMS normative scores of athletes from various sport disciplines, plotted against college student norms originally obtained by McNair in 1971 . Specifically, athletes have been found to account for significantly higher Vigor, whereas all other (negative) dimension scores remain below mean values for non-athletes, i.e., “under the surface.” This distinctive profile has been proposed to indicate greater mental health and reduced mood disturbances in athletic subjects compared to sedentary populations. By plotting individually obtained values against normative data, this specific pattern of higher positive mood and mental health in our participants compared to normative data of sedentary subjects could therefore be assessed.The psychometric assessment of mood states in our subjects was further analyzed by comparison with normative data for an athletic sample . In addition, differences in weather conditions between editions were assessed with One-Way RM ANOVA. As all participants reached the first checkpoint, we could implement a direct comparison between the two groups regarding PRE and D1 by applying two-way repeated measures ANOVA (Two-Way RM ANOVA), after normality was passed. Psychometric assessment of mood states was further analyzed through comparison with normative data for athletic samples. In accordance with Terry  with normative data obtained in athletes directly pre-competition, as well as with normative data obtained in athletes post-competition, was performed by application of unpaired Student's t-Test. Correlations between HRV indices and psychometric measurements, as well as correlations between HRV indices or psychometric measurements and performance were assessed with Pearson Product-Moment-Correlation or, if normality was not passed, Spearman Correlation. All statistical analyses were performed using SigmaPlot 12.3 . The significance level was set at p < 0.05.Data are reported as means ± standard deviations (m ± SD), if not otherwise stated. From 27 enrolled competitors, we included 16 in the statistical analysis . As a result of the extreme conditions of the competition, several participants withdrew before course completion (see section Performance). Therefore, after the race, the entire sample of all participants (ALL) was divided into two subgroups: finishers (FIN: measurements throughout the race until POST) and non-finishers (NON: measurements until D1) . This shows that the participants, who were the fastest both in the first and in the last part of the race, also accounted for the best overall time at the end of the race. Such correlations were not found for the split time between D1 and D2.Of the 16 participants included in the statistical analysis (ALL), 10 successfully completed the course (FIN). Due to general fatigue, cardiovascular distress or gastrointestinal problems, as well as injuries , 6 withdrew from the competition at earlier points (NON). Baseline anthropometric characteristics of the two subgroups, based on the completion of the race, are presented in Table max. A similar approach was used to define the quality of rest during the actual race, i.e., the time spent at checkpoints was excluded. Results of ExHR (mean HR during exercise as percentage of HRmax) and RestHR , classified according to the 4 segments mentioned above, i.e., D1a (36 h after race start), D1b (24 h before arriving at D1), D2a (24 h before reaching D2), D2b , are displayed in Table A total of 185 recordings fulfilled the criteria for analytic inclusion. They were obtained in 15 participants, as one competitor of the 2017 race belatedly volunteered to participate in the study and could not be equipped with the measuring device anymore. As reported above, recorded HR was used as a marker of exercise intensity, by normalizing absolute values with respect to the calculated HR2 and NON: −3,699.6 ms2), but the decrease was only significant (p 0.02) in NON. In fact, in NON the decrease in parasympathetic drive was to some extent greater when compared to FIN, as indicated by DFAα1, which was significantly higher at D1 only in NON  and −18.1 ms in FIN (ns) and log HF: −0.8 ms2 (p 0.04) in NON and −0.5 ms2 (ns) in FIN.For all 16 participants, a total of 53 R-R interval recordings were available and exhibited sufficient quality for analysis and assessment of HRV. 16 recordings corresponded to PRE, 13 to D1, and 10 to both D2 and POST. Morning HR pre-, post- and in-race (at checkpoints) is depicted in Figure r −0.82 p 0.02), and NN50-  with respect to HR. This could not be detected in NON.Only in FIN, a significant negative correlation between HR and mean running velocity, as well as between HR and pNN50+, was detected at PRE Figure . The negPsychometric measurements were performed in competitors of the 2015 and 2017 races, so that a total of 45 assessments  were included in the statistical analysis. Results of the POMS questionnaire revealed significant decreases in POMS Vigor and Tension, associated with an increase in Fatigue and POMS Total scores  and Borg TQR were detected in NON only, so the higher the vagal tone, the higher the TQR score. Moreover, at PRE, again in NON only, a negative correlation between POMS Fatigue and pNN50+  was observed, which indicates that the lower the vagal tone, the higher POMS Fatigue. On the other hand, in FIN at D1, a negative correlation was observed between HR and KSS Departure , indicating that the lower the HR, the higher the KSS Departure score; this was associated with a positive correlation between both pNN50–  and NN50–  and KSS Departure, which confirmed that a lower HR and a higher vagal drive were coupled with higher sleepiness; additionally in FIN at D1, there was a positive correlation between Borg TQR and DFAα2 : the higher the TQR score, so the quality of recovery after rest, the higher the DFAα2.Nevertheless, at PRE, positive correlations between several indices of vagal tone , the distribution of measurements sessions , reflected decrements in performance, while the need of rest increased. On the other hand, the continuous decrease in RestHR may also indicate higher quality of recovery  ultra-endurance exercise performance  on influencing autonomic cardiac modulation and psychometric aspects: strenuous exercise, living outdoors in subarctic winter and sleep deprivation/disturbances.In line with Millet , remaining, however, significantly above baseline values. At POST, HR was significantly lower than at D1  and not different from PRE. This would demonstrate that successful competitors  were able to positively adapt by recovering toward baseline conditions. This specific trend of HR can also be compared with observations in functional overreaching training interventions, where, initially, the increased training stimulus promotes a decrease in vagal drive , but, due to optimal adaptation, this is subsequently recovered  with respect to D1 was found  paired with higher Borg TQR values at D2 compared to D1 . This reflects a trend of increase in psychological wellbeing. As at POST, POMS Total was similar to PRE values, but significantly higher than at D1 and D2, successful recovery of mood disturbance in FIN is accentuated.Moreover, during the first part of the race, as mentioned above, the increased HR depicted the inability of participants to recover completely. However, this event was not reported by data of the Borg RPE scale Figure , which, Results of Borg TQR in NON showed a significant correlation between vagal indices and TQR scores at PRE, which may suggest that the higher the parasympathetic tone, the higher the perceived quality of recovery, underlining previous findings about the effect of parasympathetic tone on perceived fatigue in athletes , and the intervention of psychological aspects could be hypothesized (see above). All in all, assessment of HRV and psychological profile may contribute to monitor and partly predict performance in such extreme environments.Given that this is an in-field study in extreme environments, a number of possible limitations must be addressed.First of all, the sample size of 16 may appear small, however, considering that a total number of only 78 athletes competed in the three investigated editions and 27 of them enrolled in our study, we regard this number to be quite considerable and sufficient under these specific conditions.Moreover, there is a substantial difference in the distance between the in-race checkpoints  selected to perform measurement sessions and a study protocol over three equispaced checkpoints may have been favorable. However, the choice of measurement implementation was due to essential practical concerns, as previously mentioned . These concerns also held essential importance for standardizing as much as possible measurement conditions, , especially regarding HR beat-to-beat recordings.Furthermore, we aimed to allow comparison of additional data from HR continuous measurements with HRV and psychometric parameters obtained at measurement points. Therefore, continuous HR recording data were clustered and were split up in the above-mentioned four sections (see section Performance Assessment and Heart Rate Continuous Recordings).The main findings of this study are: (i) the extent of the early vagal withdrawal, associated to the timing and potential of its recovery, is crucial for success in this specific competition, (ii) a pre-competition lower resting HR, coupled with a higher vagal tone, would predict a better performance, as already reported in the literature for endurance sports (Gratze et al., LR and MM contributed equally to the study by writing the manuscript and analyzing the data. MS designed, planned and implemented the study, secured funding sources, and performed measurements and data collection. AS assisted with the measurements and data collection. AR-R, LR, and MM performed statistical analyses. RC and H-CG contributed to the study design, provided expertise and feedback. LR formatted, and, with assistance of MM and MS, revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Purpose. To present a computer-assisted-design/computer-assisted-manufacturing (CAD/CAM) technique for the design, fabrication, and clinical application of custom-made synthetic scaffolds, for alveolar ridge augmentation. Methods. The CAD/CAM procedure consisted of (1) virtual planning/design of the custom-made scaffold; (2) milling of the scaffold into the exact size/shape from a preformed synthetic bone block; (3) reconstructive surgery. The main clinical/radiographic outcomes were vertical/horizontal bone gain, any biological complication, and implant survival. Results. Fifteen patients were selected who had been treated with a custom-made synthetic scaffold for ridge augmentation. The scaffolds closely matched the shape of the defects: this reduced the operation time and contributed to good healing. A few patients experienced biological complications, such as pain/swelling (2/15: 13.3%) and exposure of the scaffold (3/15: 20.0%); one of these had infection and complete graft loss. In all other patients, 8 months after reconstruction, a well-integrated newly formed bone was clinically available, and the radiographic evaluation revealed a mean vertical and horizontal bone gain of 2.1 ± 0.9 mm and 3.0 ± 1.0 mm, respectively. Fourteen implants were placed and restored with single crowns. The implant survival rate was 100%. Conclusions. Although positive outcomes have been found with custom-made synthetic scaffolds in alveolar ridge augmentation, further studies are needed to validate this technique.  The rehabilitation of partial and total edentulism using dental implants is today considered a successful treatment procedure, with very high survival and success rates –3.However, it is frequently the case that the available bone is not sufficient for a direct implant insertion. Maxillary/mandibular bone defects are rather frequent, as a result of different processes such as tooth loss, periodontal disease, trauma, and tumours , 5. In aEven if the literature has reported successful long-term results with the use of short , 7 and tMany different techniques have been developed in order to reconstruct alveolar ridge defects and therefore allow the correct 3D insertion of dental implants –14. AmonAlthough all these surgical techniques can be successful in regenerating bone, the incidence of failures and problems that occur during these augmentation procedures is rather high , 5, 9–13The most predictable material for regeneration of the dentoalveolar process is autogenous bone, due to its peculiar properties: it is in fact osteoinductive, osteoconductive, and inherently osteogenic . HoweverAn ideal bone substitute should have excellent osteoconductivity: in fact, it should be capable of guiding the growth and proliferation of osteoblasts onto its surface , 17. IdeRecently, the use of scaffolds made of synthetic alloplastic materials has gained attention . Since tAn ideal bone substitute should easily fit into the receiving site, with a perfect shape, obtained with simple procedures , 21. WitWith the development of new digital technologies, it is now possible to analyse the bone defects in 3D and to customize bone grafts that fit perfectly into the receiving site –22. In fThe aim of the present retrospective clinical study is to report on the clinical and radiographic outcomes of bone reconstruction procedures performed with custom-made synthetic bone grafts, in three different clinical centres.The data of the patients considered for inclusion in the present retrospective clinical study came from the dental records of two different private clinics  and from the database of the dental clinic of the Insubria University . Inclusion criteria for this retrospective study were patients with a single tooth gap in the anterior/posterior maxilla or mandible, with a residual bone width of 3-4 mm, associated with a 3-wall bone defect. All these patients needed bone augmentation, prior to allowing the proper placement of dental implants and the fabrication of a functional and aesthetically acceptable implant-supported restoration. Exclusion criteria were the presence of active periodontal disease or active infection at the surgical site; poor oral hygiene or hygienic compliance; heavy cigarette smoking (>15 cigarettes/day); treatment with bisphosphonates  and any medical/general condition that could contraindicate surgery . All patients received full information about the risks related to the treatment procedure and therefore signed an informed consent form. The local ethics committee approved the present study, which was conducted in accordance with the Declaration of Helsinki on experimental studies involving human subjects, as revised in 2008.A careful assessment of the oral hard and soft tissues was performed on each patient. Panoramic and periapical radiographs were the primary investigation; after that, each patient underwent a CBCT examination, with a modern scanner . Different fields-of-view (FOV) were selected, according to the clinical indications. CBCT datasets of the partially edentulous ridges, acquired in the DICOM  format, were then uploaded onto a proprietary 3D reconstruction software  where bone segmentation was carefully performed, using thresholding tools. A virtual model of the partially edentulous ridge was therefore obtained, where the bone defect was clearly visible, and a first drawing of the scaffold could be performed Figures ; both thThe 3D virtual model of the custom-made scaffold was imported into a proprietary computer-assisted-manufacturing (CAM) software  and used to generate a set of tool-paths for fabrication by a proprietary CNC milling machine . A synthetic micro-macroporous biphasic calcium-phosphate (BCP) block, consisting of 70% beta-tricalcium-phosphate and 30% hydroxyapatite  was selected as the material of choice for the fabrication of the custom-made scaffolds. The block was therefore placed into the CNC milling machine, and milled into the size/shape of the 3D virtual model, so that a custom-made synthetic BCP scaffold was fabricated Figures . The cusAfter the injection of local anaesthesia, a full-thickness flap was elevated. The main incision  was connected with two deep, lateral releasing incisions, in order to properly view the area of the defect. The bony architecture, and consequently the bone defect, was fully exposed. A series of small perforations, 1-2 mm deep, were made on the defect walls, in order to increase the amount of bleeding at the surgical site . The cusThe sutures were removed 8–12 days after the surgery. An undisturbed 8-month healing protocol was strictly followed by all patients. During this healing period, the patients were not allowed to use any temporary removable partial denture, in order to avoid any possible compression on the regenerated area. Eight months after the regenerative surgery, the surgical site was exposed again, through the elevation of a full-thickness flap. The mini-screw used for fixation was removed, and the regenerated site showed an increased bone thickness with a considerable amount of new, well-integrated bone Figures . The surThe main clinical and radiographic outcomes of the present study were vertical/horizontal bone gain, any biological complication occurring after bone reconstruction, and implant survival. All these outcomes were carefully checked, in all patients, 6 months after implant placement and at each subsequent follow-up appointment.The vertical and horizontal dimensions of the alveolar ridge were measured in the CBCT sections, before and 8 months after the reconstructive surgery, in mm. Before reconstructive surgery, one first linear measure was taken at the future implant location. Eight months later, immediately after the placement of the implants, the same measures were repeated at the same location. These second measures were registered; then the vertical and horizontal bone gain were calculated as the difference between the second and first measurements.At each follow-up control appointment, the single crowns were removed and the stability of the implants was tested. An implant was classified as a surviving implant if still in function, without any problem, at the last follow-up control. Conversely, absence of osseointegration with implant mobility, progressive marginal bone loss due to bacterial tissue invasion (peri-implantitis), and severe marginal bone loss in the absence of symptoms/signs of infection were the conditions in which an implant was considered failed and had to be removed. before the implant placement, such as pain or discomfort after reconstructive surgery, edema, swelling, intra- or extra-oral contusion, early scaffold exposure and infection, with partial/complete loss of the graft) and late complications . With regard to late biological complications, peri-implant mucositis was defined as an inflammation of the soft tissues around the implant, with pain/discomfort and swelling, but in absence of peri-implant bone loss [The biological complications were divided into early complications  were calculated for quantitative variables, such as patient age, and gain in vertical and horizontal dimensions of the alveolar ridge. Absolute and relative frequency distributions were calculated for qualitative variables, both patient-related  and implant-related . The incidence of early and late biological complications  as well as the implant survival rate was calculated and expressed as percentages. All computations were carried out inside a dedicated datasheet .Fifteen patients  were selected for the present clinical retrospective study. Among these, four were smokers (4/15: 26.6%). All patients had been treated with a custom-made synthetic scaffold over a 7-year period, between January 2007 and January 2014; therefore the follow-up varied from 2 to 8 years (with a mean follow-up time of 4.7 years). In all patients, the regenerative surgical procedure went well. In fact, the custom-made synthetic scaffolds perfectly fitted in the bone anatomy and were therefore easily adapted to the bone defects during surgery, secured by titanium mini-screws. This excellent matching of the size/shape helped the surgeon to reduce the operation time. The healing period was uneventful for 10 patients. Five patients, however, experienced early biological complications. In fact, two of these patients (2/15: 13.3%) had mild pain and slight edema/swelling in the first week after surgery. These light symptoms/signs disappeared within two weeks. However, in the other three patients (3/15: 20.0%), early exposure of the custom-made synthetic bone graft occurred, 1, 3, and 5 months after the reconstructive surgery, respectively. These early exposures forced the surgeon to open a new full-thickness flap and to remove part of the synthetic scaffold, with the aid of a piezo-electric device. The surface of the graft was carefully cleaned, and the flap was sutured over it. All these patients were asked to apply 1% chlorhexidine gel, 2 times per day, over the site and were instructed to rinse with 0.12% chlorhexidine, 3 times per day, for a period of 1 week. After this treatment, two of the exposures were resolved with complete reepithelization of the areas and soft tissue closure, in a period of between 2-3 weeks: these early exposures did not prevent proper graft incorporation and consolidation into native bone. However, one of the exposures (1/15: 6.6%) could not be solved and determined the infection and complete loss of the graft, 5 months after surgery, in a 59-year old male smoking patient. In all the 14 remaining patients, after the 8-month healing period, a newly formed, well-incorporated bone was observed, completely filling the bony defects and therefore allowing the placement of an implant in the proper position. After 8 months, the CBCT evaluation revealed a mean vertical bone gain of 2.1 ± 0.9 mm  combined with a mean horizontal bone gain of 3.0 ± 1.0 mm . In total, 14 implants were successfully placed . All these implants (14/14: 100%) were restored with single crowns. No further (late) biological complications were reported, and an implant survival rate of 100% (14/14 surviving implants) was found.The use of implants for supporting dental prostheses is continuously expanding and it is estimated that the market will have the same trend in the future –3.One of the main limitations of the implant treatment is the unavailability of adequate bone support, mainly caused by periodontal disease, but also as a result of tooth agenesis, traumatic injuries, or other lesions  , 5.Many different surgical reconstructive techniques have been introduced in order to reestablish an adequate bone volume and allow proper implant placement –14: amonAutogenous bone is still considered the material of choice in bone augmentation procedures . HoweverMore recently, synthetic bone grafts  have been introduced, in order to overcome these limitations , 33–35. Blocks of synthetic biomaterials are already available in the market. However, these blocks are prepared in preformed size/shapes and need to be adapted to the patient's bone defect during the surgery , 23, 24.Nowadays, modern digital technologies allow the surgeon to virtually design and then fabricate custom-made synthetic porous scaffolds, for use in bone reconstructive procedures –26, 36. The CAD/CAM procedure for the fabrication and application of custom-made synthetic scaffolds can be divided into three different steps: the virtual planning and design of the scaffold, the milling of the scaffold into the exact size/shape from a preformed synthetic bone block, and, finally, the reconstructive surgery , 24, 26.Jacotti et al.  reportedIn our present study, we have reported on the clinical and radiographic outcomes obtained with this innovative, CAD/CAM procedure for alveolar ridge augmentation. From a surgical point of view, the custom-made synthetic scaffolds were of satisfactory size, shape, and appearance; they matched the defect area, suited the surgeon's requirements, and were easily implanted. This perfect match contributed to reducing the time for surgery and to the good healing of the bone defect. Only a limited number of patients experienced biological complications, such as pain/swelling (2/15: 13.3%) and exposure of the scaffold (3/15: 20.0%); one of these patients, however, experienced infection of the scaffold and complete graft loss. In all other patients, 8 months after reconstruction, a well-integrated newly formed bone was clinically available, and the CBCT evaluation revealed a mean vertical and horizontal bone gain of 2.1 ± 0.9 mm and 3.0 ± 1.0 mm, respectively. Fourteen implants were placed and restored with single crowns. The implant survival rate was 100%.The present CAD/CAM technique for the fabrication of custom-made synthetic scaffolds for alveolar ridge reconstruction undoubtedly has varied benefits: in fact, the accurate reproduction of the patient's anatomy helps to reduce the time needed for the surgical procedure and therefore the morbidity and risk of infection for the patient , 24, 26.However, this procedure has limitations. As reported by the current literature, for a successful alveolar ridge augmentation it is necessary to achieve a perfect fit of the bone block, a precise stabilization of the graft but also a well vascularized bone bed , 21. TheAlthough the procedure for the design, fabrication, and clinical application of custom-made synthetic bone grafts described here presents the aforementioned limitations, our present positive clinical and radiographic outcomes seem to suggest it as a possible alternative to conventional surgical techniques, such as alveolar ridge augmentation with onlay/inlay autogenous bone blocks , 21, 26.In the present retrospective clinical study, we have presented an innovative CAD/CAM technique for the design, fabrication, and clinical application of custom-made synthetic bone grafts, for alveolar ridge augmentation. Although positive clinical and radiographic outcomes have been found in this study, with an excellent fit of the scaffolds during surgery and a well-integrated newly formed bone clinically available 8 months after bone reconstruction, a rather high incidence of biological complications, such as early graft exposure (20%), were reported. Further studies with a larger patient sample and an appropriate design  are therefore needed to draw specific conclusions about the reliability of the present technique and to confirm our positive clinical and radiographic outcomes.